Screening and identification of emodin as an EBV DNase inhibitor to prevent its biological functions

TW01 is an Epstein-Barr virus-negative NPC cell line [34]. NA is an EBV-positive NPC cell line that is derived from the EBV infection into TW01 cells [9]. NPC cell lines were cultured in Dulbecco's modified eagle medium supplemented with 10% foetal bovine albumin with (NA) or without (TW01) 400 μg/mL G418 (Sigma-Aldrich, St. Louis, MO, USA).

Emodin, 12-O-tetradecanoyl-phorbol-1,3-acetate (TPA), and sodium butyrate (SB) were purchased from Sigma-Aldrich. PicoGreen was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The EBV DNase antibody used in this study was anti-DNase 311H [52].

To express recombinant EBV DNase and mutants in E. coli, the plasmid encoding the wild-type EBV DNase BGLF5 sequence (P3HR1 strain, 470 amino acids) was subcloned into the vector pET-15b [35]. Mutants were generated using site-directed mutagenesis strategies established previously [37]. Mutants with the E225A single mutation (mut E225A) or E225A/K227A double mutations were generated by site-directed mutagenesis and subcloned into pET-15b [37].

To express EBV DNase in NPC TW01 cells, BGLF5 sequences were subcloned into the pEGFP-CPO-IRES-puro vector, which was derived from pEGFP-C1 by CPO site ligation [56].

Expression and purification of EBV DNase were performed as described previously [38]. Briefly, pET-15b-driven EBV DNase or mutants were transformed into E. coli BL21(DE3) pLysS. At OD600 0.4 ~ 0.6, IPTG (1 mM) was added to the culture for 2 h. Cells were harvested by centrifugation, and cell pellets were resuspended in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8.0, 0.1% NP-40 and 10% glycerol). After sonication and centrifugation at 39 000 g for 20 min, the supernatant was used as the starting material for DNase purification, and subjected to a His-Bind column (Novagen, Pretoria, South Africa). The column was washed sequentially with two times of wash buffer 1 (20 mM Tris/HCl, pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole), two times of wash buffer 2 (20 mM Tris/HCl, pH 8.0, 10% glycerol, 50 mM imidazole), and five times of elution buffer with increasing imidazole (20 mM Tris–HCl, pH 8.0, 10% glycerol, 100 ~ 500 mM imidazole). The purified proteins were analysed by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE) (Additional file 1: Figure S1), quantified with a Bradford assay kit (Bio-Rad, Hercules, CA, USA), and then stored at − 70 °Ϲ until future use.

For DNA cleavage assay, 500 ng of purified EBV DNase mixed with 0.5 mg of the plasmid DNA pEGFP-C1 (Invitrogen) in reaction buffer (50 mM Tris/HCl, pH 8.0, 4 mM b-mercaptoethanol and 4 mM MgCl₂) incubated at 37 °C for 1 h. The mixtures were analysed by agarose gel electrophoresis.

In the isotope-based method, AN activity was measured by assessing the release of soluble nucleotides from isotope-labelled DNA (Additional file 1: Figure S2a) [29]. To perform the radioactivity-based assay, C¹⁴-labelled calf thymus DNA was used as the DNA substrate. Purified recombinant proteins of wild-type and mutant EBV DNase (1 μg) were diluted with dilution buffer (50 mM Tris–HCl, pH 8.0, 10 mM MgCl₂, 10 mM 2-mercaptoethanol, 0.05% bovine serum albumin), and then were incubated with 1 μg C¹⁴-labelled DNA for 1 h at 37 °Ϲ. After adding the stop solution (50% TCA and sheared calf thymus DNA), the sample was spun down and the radioactivity of supernatant was measured using a liquid scintillation counter (LS-6000; Beckman, Fullerton, CA, USA). Enzyme activity was defined as the percentage of DNA hydrolysed, which was calculated as the count per minute (cpm) of each sample normalised to the total amount of isotope usage.

For the PicoGreen-labelled method, AN activity was assessed using PicoGreen-incorporated double-stranded DNA, as described in previous studies (Additional file 1: Figure S2b). Briefly, purified recombinant proteins (1 μg) were diluted with dilution buffer (50 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.05% BSA). The diluted solutions were mixed with 90 ng salmon sperm DNA, and adjusted with various concentrations of metal ions, pH, or other conditions for 1 h at 37 °Ϲ, respectively. To determine the influence of pH, reaction buffers of pH 7–9 were prepared with 50 mM Tris–HCl, pH 9.5–10.5 with 25 mM borate buffer, and pH 11–11.5 with sodium bicarbonate-NaOH. After then, the samples were mixed with 100 μl PicoGreen dye (Molecular Probe, Waltham, MA, USA; 200-fold diluted in 10 mM Tris–HCl, pH 8.0, and 10 mM EDTA) to label DNA, then incubated the mixture of samples and PicoGreen dye for 2 to 5 min, and finally, fluorescence (Excitation: 480 nm; Emission: 520 nm) was detected using a microplate reader (Infinite M 200, Tecan, Männedorf, Switzerland). Enzyme activity was defined as the percentage of DNA hydrolysed, which was calculated as follows: the fluorescent read of total labelled DNA was subtracted from that of the fluorescent read of each sample, and the result was normalised to the fluorescent read of total labelled DNA.

For screening natural compounds against AN activity, purified recombinant proteins (1 μg) were diluted with a dilution buffer. The diluted solutions were mixed with 90 ng salmon sperm DNA and indicated amount of natural compounds for 1 h at 37 °Ϲ. After then, the samples were mixed with 100 μl PicoGreen dye as the same as described above. The fluorescence (Excitation: 480 nm; Emission: 520 nm) was detected using a microplate reader (Infinite M 200, Tecan, Männedorf, Switzerland). The IC50 value of each compound was calculated by the Microsoft Excel or SigmaPlot using the linear regression.

For TW01 transfection, plasmid was transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Briefly, TW01 cells were plated for 24 h. Before transfection, the plasmid DNA was mixed with Lipofectamine 2000 in Opti-MEM medium (Invitrogen) and incubated for 20 min, then subjected to the culture well containing TW01 cells.

MN formation was detected as described previously [25, 36, 56]. Briefly, TW01 cell lines transfected by lipofectamine 2000 (Invitrogen) with EBV DNase-expressing plasmids or parental vectors pEGFP-CPO-IRES-puro were plated on cover slides, with or without emodin treatment. After 24 h of incubation, the cells were washed with phosphate-buffered saline (PBS) (pH 7.4) and fixed with cold methanol for 15 min. After washing with PBS, the cells were stained with Hoechst dye (0.2 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 15 min. The MN was counted under a fluorescence microscope. The percentage of MN formation was defined as the number of MN divided by the total number of counted cells; at least 1000 cells were counted in each group. The IC50 value of each sample was calculated by the Microsoft Excel or SigmaPlot using the non-linear regression.

Western blotting was performed as described previously [38]. Briefly, purified EBV DNase and its mutants were subjected to SDS–PAGE. The proteins were transferred to Hybond-C membranes (Amersham Biosciences, Amersham, United Kingdom). The membranes were blocked with 4% milk for 1 h and incubated with 1:1 diluted EBV DNase antibodies [52] overnight at 4 °Ϲ. After washing with the wash buffer (10 mM Tris–HCl, pH 8.0, 0.9% NaCl), the membranes were incubated with a secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG, Amersham, diluted 1:2500) and developed using a developing substrate (Amersham Biosciences Ltd.). Luminescence was detected by exposure to an X-ray film.

Comet assay was used to detect DNA damage [56], according to the manufacturer’s instructions (Trevigen, Minneapolis, MN, USA). Briefly, the cells were transfected with the vector or DNase-expressing plasmids for 3 h. Next, emodin was added, and the cells were incubated for 24 h. The treated cells (1 × 10⁵/ml) were harvested and resuspended in a 1:10 (v/v) mixture of PBS and molten low-melting agarose (Trevigen, Minneapolis, MN, USA) at 37 °Ϲ for a total of 100 μl. The mixture (75 μl) was immediately pipetted onto CometSlideTM (Trevigen, Minneapolis, MN, USA). After cooling, the slides were treated with lysis buffer and subsequently with an alkaline solution (300 mM NaOH and 1 mM EDTA) to denature cellular DNA. The samples were subjected to electrophoresis and stained with SYBR Green to observe comet tails using a fluorescence microscope. One hundred comets were scored randomly. Each comet was assigned a visual score of 0–4 based on the tail length. The length and intensity of comet tails are positively correlated to the visual scores. The standards of scoring are: class 0 is a score of 0, class 1 is a score of 1, and so on. The total visual score was calculated to determine the level of DNA damage [3, 40]. The IC50 value of each sample was calculated by the Microsoft Excel or SigmaPlot using the non-linear regression.

To evaluate the production of EBV, the copy number of the EBV genome in the released particles was determined as previously described [13, 55]. Briefly, NA cells were pre-treated with emodin for 1 h prior to TPA (40 ng/mL) and SB (3 mM) treatment for 48 h. Supernatants were filtered through a 0.45 μM filter and treated with DNase I and DNase I buffer (10 mM Tris–HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH 7.6) at 37 °Ϲ for 60 min. Subsequently, EDTA (2 mM, pH 8.0) was added to terminate the reaction, and each sample was treated with proteinase K (0.1 mg/mL, sample: proteinase K = 1:1 [vol/vol]) at 50 °Ϲ for 60 min. Proteinase K was then inactivated at 75 °Ϲ for 20 min. Subsequently, real-time PCR analysis [13] was performed to detect the BALF5 fragment using the following primers: sense, 5′-CGGAGTTGTTATCAAAGAGGC-3′; antisense, 5′-CGAGAAAGACGGAGATGGC-3′. The qPCR cycling conditions were as follows: denaturation for 5 s at 95 °Ϲ, annealing for 20 s at 60 °Ϲ, and extension for 2 s at 72 °Ϲ for 45 cycles in the LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA). NA cells activated by TPA + SB without emodin were used as a positive control, whereas cells that were not treated with any compounds were used as mock controls. The relative fold change in viral production was defined as the qPCR value of each sample normalised to the individual cell numbers divided by that of the mock control. The EC50 value of each sample was calculated by the Microsoft Excel or SigmaPlot using the non-linear regression.

The protein structure of EBV DNase BGLF5 was obtained from the NCBI server (https://​www.​ncbi.​nlm.​nih.​gov/​Structure/​pdb/​2W45) [7]. The software Swissdock [23, 24] and UCSF Chimera [41] were used for predicting a possible docking model between EBV DNase and emodin. The input files were in the PDB formats. The PDB format of emodin was obtained from the PubChem server (https://​pubchem.​ncbi.​nlm.​nih.​gov/​compound/​Emodin).

According to previous studies, EBV DNase possesses a catalytic site for the degradation of DNA substrates, which is conserved in other herpesviral ANs and is homologous to that of type II restriction endonucleases [31, 38]. This core region is composed of D203…E225XK227 (D…EXK), among which, D203 is important for metal ion coordination, E225 for coordinating metal ions DNA binding, and K227 for DNA binding [38].

To establish a fluorescence-based method to detect AN activity, the purified recombinant proteins of the wild-type EBV DNase (wt EBV DNase) and its two mutant derivatives (mut E225A and mut E225A/K227A) derived from E. coli BL21(DE3) strain pLysS were used to detect nuclease activity (Fig. 1a) [37]. The protein status in the three clones was examined using western blot analysis (Fig. 1b). Nuclease activity was examined using three approaches, including DNA cleavage analysis, isotope-based assay and fluorescence-based methods. As shown in Fig. 1c, the decrease in band intensity revealed that wt EBV DNase had strong activity, whereas the mut E225A retained moderate activity, and mut E225A/K227A retained weak activity which was consistent with our previous results [37]. The results of the isotope-based assay were similar to those of the DNA-cutting analysis (Fig. 1d). Furthermore, the results of the fluorescence-based method were consistent with those of conventional approaches (Fig. 1e). These results suggest that the fluorescence-based assay was successfully developed and is ready for further examination.

The catalytic environment is important for nuclease cleavage. Therefore, we determined whether the PicoGreen-labelled method could be used to detect the effect of environmental conditions on EBV DNase activity. The following environmental factors are known to affect the nuclease activity of EBV DNase: alkaline condition (pH 8 ~ 9), 37 °Ϲ and the presence of Mg²⁺ ion [12, 30, 43]. As shown in Fig. 2a, EBV DNase exhibited maximum activity at pH 8.0, whereas the control reactions showed no detectable activity. This result fits the existing knowledge about herpesviral nucleases and is consistent with the terminology “alkaline nucleases” [12, 43].

Next, to determine the optimal temperature, reactions were carried out at the optimum pH (pH 8.0) at various temperatures. As shown in Fig. 2b, EBV had the best performance at 37.2–39.5 °Ϲ, which is close to the preferred reaction temperature of EBV DNase, 37 °Ϲ, reported in previous studies [12, 43]. Divalent metal ions, especially magnesium (Mg²⁺) and manganese (Mn²⁺) are essential for the catalysis of DNA degradation [5], including those herpesviral nucleases [30, 47, 50]. To determine the optimal concentration of divalent cations for EBV DNase using the developed fluorescence-based approach, various concentrations of Mg²⁺ and Mn²⁺ were used. The CTL group is in the same reaction condition without EBV DNase. The results showed that EBV DNase lost its activity in the absence of either magnesium or manganese, suggesting that divalent cations are required for enzymatic activity (Fig. 2c, left and right panels). In contrast, EBV nuclease activity increased with increasing magnesium concentration and maximum activity was seen at 5 mM Mg²⁺ (Fig. 2c, left panel). For manganese, EBV DNase had a preferential Mn²⁺ concentration of 50 μM (Fig. 2c, right panel). Interestingly, considering the optimal activity for DNA hydrolysis, EBV DNase hydrolyzed 88% of substrate DNA at the highest Mg²⁺ concentration tests compared to 28% at the highest Mn²⁺ concentration tests (Fig. 2c, left and right panels), suggesting EBV DNase had lower efficiency of DNA hydrolysis with Mn²⁺ compare to that with Mg²⁺.

Furthermore, ionic strength is important for herpesviral nuclease activity. KCl, NaCl and (NH₄)₂SO₄ have been reported to exhibit an inhibitory effect on herpesviral nucleases [4, 29, 50]. To determine whether these phenomena could be detected using the fluorescence-based assay, salts were added to the reaction mixtures at various concentrations. As expected, EBV DNase nuclease activity was inhibited with increasing concentrations of KCl, with an IC₅₀ value of 140 mM (Fig. 2d, left panel). Consistently, EBV DNase was resistant to high concentrations of NaCl (IC₅₀ = 125 mM) (Fig. 2d, middle panel). In contrast, with (NH₄)₂SO₄, EBV DNase was inhibited significantly, with an IC₅₀ value of 15 mM (Fig. 2d, right panel). These results indicate that the fluorescence-based assay can determine the sensitivity of EBV DNase to high ionic strengths.

Taken together, these results suggest that the fluorescence-based nuclease activity assay can be used to detect the preferred environmental conditions for EBV DNase activity.

Nuclease inhibitors have potential clinical applications [18], especially in anti-viral therapy [1, 6, 8, 32]. Therefore, we tested several natural compounds using this fluorescent-based platform to identify inhibitors of EBV DNase. As shown in Fig. 3a, emodin significantly inhibited the AN activity of EBV DNase (IC50:31 μM). Kaempferol and epigallocatechin gallate (EGCG) exhibited moderate inhibition (IC50: 69 and 89 μM, respectively), whereas sulforaphane (SFN) and diallyl disulfide (DADS) exhibited poor inhibition of EBV DNase (IC50: > 100 μM) (Fig. 3a). To verify these results, emodin and SFN were further tested using the conventional DNA cleavage assay. As shown in Fig. 3b, EBV DNase was inhibited by emodin, whereas SFN had a lower inhibitory effect on AN activity, which was consistent with the results of the fluorescence-based assay.

Taken together, these results suggest that the fluorescence-based assay can be used for screening AN inhibitors and that emodin is a potential target of EBV DNase inhibitors.

EBV DNase has important effects on cellular processes and the viral life cycle. It can cause DNA damage and induce genomic instability [56], and plays an important role in viral genome egress and virion production [20]. The comet assay is a standard protocol for detecting DNA damage, and MN formation is an indicator of genomic instability [26]. Next, we performed these two assays to determine the effect of emodin on EBV DNase-induced DNA damage and genomic instability. At first, emodin has been shown a cytotoxic effect on the NPC TW01 and NA cell lines for 48 h treatment. The CC50 values of emodin on TW01 and NA cell lines were 50 and 89 μM, respectively (Fig. 4a). Based on these results, we chose 1 to 50 μM of emodin as the working concentrations for the following experiments. And then NPC TW01 cells were transfected with an EBV DNase-expressing plasmid or vector for 3 h. Then the cells were treated with various concentrations of emodin. After 24 h of incubation, the cells were harvested for comet assay and MN formation. After checking the expression of EBV DNase in each treating group (Additional file 1: Figure S3), the EBV DNase-transfected group without emodin treatment displayed the highest visual score; the comet index revealed robust DNA damage compared to the vector control (Fig. 4b,). However, emodin treatment reduced the visual scores. These results suggest that emodin treatment decreased the comet index in the EBV DNase group, but not in the vector group (Fig. 4b). A similar trend was observed in the MN formation assay. EBV DNase expression induced significant MN formation; however, this phenomenon was repressed by emodin treatment (Fig. 4c). Furthermore, we determined whether emodin treatment inhibited viral production. As shown in Fig. 4d, viral production decreased with increasing emodin concentrations. We further determine whether emodin affects Taq polymerase activity in a PCR reaction. The culture media with 48 h activation were collected and treated as the same as the procedure of viral DNA detection, following with adding for various amounts of emodin to detect EBV BALF5 fragment. The results revealed that the detection values were not affected significantly, suggesting emodin did not inhibit Taq activity in the detection of viral production (data not shown).

These results suggest that emodin can not only inhibit EBV DNase-induced DNA damage and genomic instability but can also inhibit EBV production.

The catalytic site of the EBV DNase was identified as D….EXK motif at positions 203 and 225–227 of the protein sequence, which is responsible for DNA and metal ion binding [38]. We further performed a computational analysis to predict a docking model of EBV DNase with emodin. Because the protein structure of EBV DNase has been resolved [7](PDB ID: 2W45), using Swissdock [23, 24] and UCSF Chimera [41], we predicted a possible docking model in which emodin occupied the catalytic cavity formed by D203, E225, I226, and K227 (Fig. 5). This hypothetical model predicts the possible sites at which emodin interacts with EBV DNase and provides a useful tool to identify other nuclease inhibitors.