Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene

Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified by mass spectrometry. The results of RT-PCR, Western blotting, and pharmacological inhibition tests revealed that Us5 is transcribed mainly in the late stage of viral replication. The IFA results revealed that Us5 was localized throughout DPV-infected cells but was localized only to the cytoplasm of transfected cells. Mass spectrometry and Western blot analysis showed that Us5 was a virion component. Next, to study the antiapoptotic function of DPV Us5, we found that DPV CHv without gJ could induce more apoptosis cells than DPV-CHv BAC and rescue virus. we constructed a model of apoptosis in duck embryo fibroblasts (DEFs) induced by hydrogen peroxide (H2O2). Transfected cells expressing the Us5 gene were protected from apoptosis induced by H2O2, as measured by a TUNEL assay, a caspase activation assay and Flow Cytometry assay. The TUNEL assay and Flow Cytometry assay results showed that the recombinant plasmid pCAGGS-Us5 could inhibit apoptosis induced by H2O2 in DEF cells. However, caspase-3/7 and caspase-9 protein activity upregulated by H2O2 was significantly reduced in cells expressing the recombinant plasmid pCAGGS-Us5. Overall, these results show that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized in the cytoplasm, and can inhibit apoptosis induced by H2O2 in DEF cells.

mechanism of Us5 is not clear. Based on the past experimental results, we just know that Us5 protein can regulate caspases, cause mitochondrial membrane potential decline, and promote the production of reactive oxygen species (ROS) 18,21 .
Apoptosis is an important mechanism of host immune defense. The apoptotic process is mainly characterized by cell shrinkage, chromatin aggregation, and apoptotic body formation. Thus far, apoptosis has been shown to be induced by two classical pathways: the extrinsic and intrinsic apoptotic pathways. Caspases are cysteine proteases that are extremely important for intracellular apoptotic pathways, and caspase-9 and caspase-8 are involved in the intrinsic and extrinsic apoptotic pathways, respectively; both caspase-8 and caspase-9 activate the downstream molecule caspase-3 to initiate apoptosis. H 2 O 2 is an apoptosis inducer that causes cells to undergo oxidative stress and increases intracellular ROS. ROS reduce the mitochondrial membrane potential and activate caspase-9, which subsequently activates the downstream molecule caspase-3 22 .
Our laboratory has previously demonstrated that the function of DPV Us5 is slightly impaired in viral replication, virion assembly and cell-to-cell spread and that is not essential in virion envelopment 23 . However, information regarding the DPV Us5 gene is limited. In this study, we further performed a molecular characterization and investigated the antiapoptotic function of the DPV Us5 gene. The results of this study will provide a foundation for studying the pathogenesis of DPV.

Results
Kinetics of DPV Us5. The melting curves showed that the specificities of the primers were excellent, and standard curves were established to evaluate the efficiency of the assays (data not shown). The results showed that the Us5 gene transcript was detected at 6 hours post infection (hpi); Us5 expression gradually increased, peaking at 36 h, and then steadily decreased at 48 hpi, 54 hpi, and 60 hpi (Fig. 1A). As shown in Fig. 1B, a specific protein band of approximately 130 kDa was initially detected at 24 h. Peak expression occurred at 36 hpi, decreasing steadily thereafter, and Us5 was still detected at 60 hpi. The gene and protein expression levels of DPV Us5 are consistent with those of a late gene. To further verify the Us5 gene type, the protein synthesis inhibitor   (Fig. 5A). Compared to those of untreated cells, the nuclei of H 2 O 2 -treated cells were smaller and distorted; in addition, chromatin was collected on the nuclear membrane and apoptotic bodies appeared (Fig. 5B). Positive TUNEL staining, a hallmark of apoptotic cell death, was detected in H 2 O 2 -treated cells, whereas non-H 2 O 2 -treated cells were negative for TUNEL staining (Fig. 5C). In addition, Flow Cytometry results indicated that H 2 O 2 -treated cells have 22% apoptosis cells (Fig. 6C). H 2 o 2 activates caspase-3 and caspase-9 in DEF cells. The mRNA level of caspase-3 in H 2 O 2 -treated cells was approximately 1.65 times that in untreated cells, and the mRNA level of caspase-9 in H 2 O 2 -treated cells was approximately 3 times that in untreated cells (Fig. 6A). H 2 O 2 treatment of cells led to a significant increase in the activity of the caspase-3/7 and caspase-9 proteins (Fig. 6B). The luminescence values of caspase-3/7 and caspase-9 in untreated cells were 4.5 × 10 3 RLU and 3.0 × 10 3 RLU per ten thousand cells, respectively. The luminescence value of caspase 3/7 and caspase 9 in treated cells were 6.0 × 10 3 RLU and 8.0 × 10 3 RLU per ten thousand cells, respectively (Fig. 6B).

Expression of the recombinant plasmid pCAGGS-Us5 in DEF cells. pCAGGS and pCAGGS-Us5
plasmids were transfected into DEFs. In Fig. 7A, fluorescence is indicated in green (FITC) in pCAGGS-Us5 plasmid-transfected cells. The expression of the pCAGGS-Us5 plasmids was detected by Western blotting, which showed approximately 130 kDa bands (Fig. 7B). Similarly, the expression of DPV Us5 was detected, and the Western blot showed approximately 130 kDa bands (Fig. 7C).   (Fig. 8A). The results showed that the caspase-3/7 protein activity in H 2 O 2 -treated cells transfected with the pCAGGS plasmid was approximately 6 × 10 3 RLU per ten thousand cells, while that of H 2 O 2 -treated cells transfected with the recombinant plasmid pCAGGS-Us5 was approximately 3.3 × 10 3 RLU per ten thousand cells (Fig. 8B). The results also showed that the caspase-9 protein activity of

Anti-apoptosis ability of DPV CHv-BAC parental viruses, DPV CHv-BAC-ΔgJ mutant viruses and CHv-BAC-ΔgJR rescue viruses.
According to the following results, we further explored whether Us5 could inhibit apoptosis induced in DPV infected cells. There have been reported that DPV CHv can cause DEF cells apoptosis 24 , and we constructed the DPV CHv-BAC-ΔgJ mutant viruses successfully 23 . Therefore, the DEF cells were infected with DPV-CHv, DPV CHv-BAC-ΔgJ mutant viruses and DPV CHv-BAC-ΔgJR rescue viruses respectively. The apoptosis DEF cells of recombination viruses relative to the parental virus were investigated at 48 hpi utilizing DAPI staining. As is shown in the Fig. 9A, the DPV CHv-BAC parental virus and the DPV CHv-BAC-ΔgJR rescue viruses induced apoptosis DEF cells that were similar in number. The apoptosis cells induced by DPV CHv-BAC-ΔgJ mutant viruses more than the parental virus. To better assess the apoptosis cells induced by different viruses, Flow Cytometry was analyzed the DEF Cells of apoptosis, this analysis also confirmed that the apoptosis cells induced by DPV CHv-BAC-ΔgJ mutant viruses more than the parental viruses ( Fig. 9B).

Discussion
Until now, reports about the DPV Us5 gene have been limited. Our group constructed two different mutated viruses, a gJ deleted mutant, ΔgJ, and a rescue virus with gJ restored, ΔgJR, showing that the gJ was slightly impaired in viral replication, virion assembly and cell-to-cell spread and was not essential in virion envelopment 23 . In this study, we further studied the function of DPV pUs5 as the first step in exploring the molecular characterization and antiapoptotic function of the DPV Us5 protein.
During herpesvirus infection, viral genes are expressed sequentially in three distinctly defined stages: immediate early gene (IE), early gene (E), and late gene (L). The IE genes are immediately transcribed upon infection, without interaction with other proteins 25 . The E genes are transcribed prior to viral DNA replication in an IE protein-dependent manner. Transcription of the L genes begins after the synthesis of DNA, and viral protein translation begins 7,26 . HSV-1 Us5 is a late gene, with partial dependency on DNA replication for its expression 21 . The current study demonstrates that Us5 is similar to other HSV glycoproteins in that it is regulated as a late gene. To identify the DPV Us5 gene type, we used Western blot and RT-PCR analyses to study the expression and transcriptional kinetics of DPV Us5 at different time points after infection. Based on our group of previous studies 27-31 , we speculated that accumulation of the DPV Us5 protein may occur during the late stage of infection. The IE genes are expressed in the presence of the protein synthesis inhibitor CHX, while the transcription of the E and L genes is inhibited in the presence of CHX. To further study DPV Us5 gene, we treated infected cells with a protein synthesis inhibitor, CHX, and a DNA polymerase inhibitor, ganciclovir (GCV), and observed that the Us5 gene was inhibited in both the CHX and GCV groups.
Intracellular viral protein localization is associated with viral protein function, but intracellular DPV Us5 localization has thus far remained unclear. A previous publication demonstrated that Us5 was located on the surface of HSV-1-infected cells. Further studies showed that Us5, similar to other HSV glycoproteins, was mainly found in intracellular membranes, especially in Golgi bodies, the endoplasmic reticulum and endosomes 21 . In this study, DPV infection in DEFs was detected by IFA. The results showed that DPV Us5 was mainly localized in the cytoplasm. Then, the recombinant plasmid pCAGGS-Us5 was transfected into DEF cells and revealed that Us5 was localized mainly in the cytoplasm. This finding indicates that Us5 modulates cellular functions occurring in the cytoplasm. DPV Us5 is a glycoprotein that may be differentially glycosylated to form different glycoproteins during posttranslational modification. The recombinant plasmid pCAGGS-Us5 was transfected into DEF cells and detected by Western blotting, which showed one specific band of approximately 130 kDa. Interestingly, the expression of Us5 after DPV infection in DEFs, as detected by Western blotting, also showed one specific band of approximately 130 kDa. The size of the Us5/gp2 protein encoded by the α-herpesvirus Us5 varies greatly among different viruses. The gp2 gene of equine herpesvirus-1 (EHV-1) is homologous to the Us5 gene and expresses www.nature.com/scientificreports www.nature.com/scientificreports/ three proteins with different molecular weights, 230 kDa, 65 kDa and 42 kDa 11 ; the Us5 and gD proteins of HSV-1 and the gG protein of Macacine herpesvirus-1 also have different molecular weights 21,32 .
Viruses infect cells to facilitate their own breeding and to ensure their long-term survival in host cells, and viruses can evolve the necessary functions to prevent apoptosis and delay cell death. This ability to evolve is one of the biological characteristics of α-herpesviruses 33 . Many studies have confirmed that α-herpesviruses encode proteins with antiapoptotic functions [16][17][18][34][35][36] . During the early stage of infection, these genes promote the survival of host cells, facilitate the replication of the virus itself and facilitate the production of subviruses. One of the functions reported for the Us5 orthologs of HSV-1 is the inhibition of apoptosis induced by viral infection www.nature.com/scientificreports www.nature.com/scientificreports/ and exogenous factors 18,37 . To study the antiapoptotic functions of DPV Us5 gene encoding protein, we first established an apoptotic model in which the apoptosis of DEF cells was induced by H 2 O 2 . There are many ways to induce cell apoptosis in vitro, such as with UV, sorbitol, and H 2 O 2 , among other methods [38][39][40][41] , and H 2 O 2 has been used to establish apoptosis models in multiple cell types for antiapoptotic function experiments [42][43][44] . The morphology of the cells changed after treatment with 400 μM H 2 O 2 . These changes are consistent with the characteristics of apoptosis 45 . In addition to examining morphology, detecting apoptotic factors is another classic way to verify apoptosis. In the apoptotic cascade, caspases, including caspase-3, are the major effectors of apoptosis 46 . Therefore, to further verify that H 2 O 2 induces DEF apoptosis, detection of the mRNA levels and activity of the apoptotic factor caspase-3 was necessary. In the current experiment, all of the data showed that the caspase-3 mRNA levels and activity were increased in H 2 O 2 -treated cells relative to those in control cells. These results prove  www.nature.com/scientificreports www.nature.com/scientificreports/ the cleavage of downstream caspase-9, which activates other caspases, resulting in apoptosis 22,[47][48][49] . In this study, the mRNA and activity levels of caspase-9 in H 2 O 2 -treated cells were significantly increased compared to those in control cells, suggesting that H 2 O 2 -induced apoptosis in DEF cells may be related to the mitochondrial pathway. This study is the first to show that DPV Us5 is necessary for the effective protection of cells against apoptosis triggered by H 2 O 2 . The results showed that the expression of the Us5 gene encoding protein inhibits apoptosis induced by H 2 O 2 and inhibits the H 2 O 2 -induced activation of caspase-3/7 and caspase-9, suggesting that the Us5 gene may inhibit the intrinsic pathway. The DPV CHv-BAC parental viruses and the DPV CHv-BAC-ΔgJR rescue viruses induces apoptosis DEF cells that were similar in number. The apoptosis cells induced by DPV CHv-BAC-ΔgJ mutant viruses more than the parental viruses. However, some studies have shown that Us5 can inhibit the extrinsic pathway. In one study, the removal of Us5 from HSV-1 significantly abolished protection against Fas-mediated apoptosis and partially reduced protection against UV radiation by inhibiting caspase-3/8 activation 50 . In addition transfecting Us5 has been found to inhibit Fas/UV-induced apoptosis and to weaken www.nature.com/scientificreports www.nature.com/scientificreports/ the activation of caspases 18 . Overall, the results suggested that Us5 is sufficient to establish an antiapoptotic phenotype. Us5-deleted HSV-1 shows a weakened protective effect against cytotoxic T lymphocyte-induced apoptosis, and gJ is sufficient to inhibit F 0 F 1 ATP synthase activity and the production of ROS to inhibit apoptosis 51 . However, the mechanism by which DPV Us5 blocks apoptosis remains elusive. Others should further study the underlying mechanism through the use of additional apoptosis models other than H 2 O 2 -induced apoptosis.
In summary, this study found that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized mainly in the cytoplasm, and can inhibit apoptosis induced by H 2 O 2 . Plasmid construction and transfection. The Us5 gene fragment was ligated into the pCAGGS plasmid by homologous recombination using a ClonExpress ® II One Step Cloning Kit. DEFs were transiently transfected in 6-well plates with 2.5 μg of the pCAGGS-Us5 recombinant plasmid using 5 μl of Lipofectamine 3000 transfection reagent (Thermo Scientific, Massachusetts, USA) according to the manufacturer's guidelines. At 48 h after transfection, the supernatants of the DEF lysates were collected and stored at −80 °C until further use. The control group included the supernatant collected from DEFs transfected with the empty pCAGGS plasmid. The expression levels of the pCAGGS-Us5 plasmids were confirmed by Western blotting. The primers were designed with software of PRIMER 5 and are listed in Table 2.

Isolation of RNA and analysis of Us5, caspase-3, and caspase-9 mRNA expression by RT-PCR.
Total RNA was extracted from DPV-infected DEFs at different time points post infection (6 h, 12 h, 24 h, 36 h, 48 h, 54 h, and 60 h). The DEFs included H 2 O 2 -treated cells and mock-treated cells as well as transfected and nontransfected H 2 O 2 -treated cells, and the total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer's instructions. Extracted RNA was immediately reverse transcribed into first-strand cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). For RT-PCR, a 20 µL total reaction volume was used that contained 10 µL of SYBR Premix Ex Taq II (Tli RNaseH Plus), 1 µL of forward primer, 1 µL of reverse primer, 6 µL of RNase-free water and 2 µL of cDNA. The thermal cycling program included an initial denaturation step for 30 seconds at 95 °C followed by 40 cycles of 5 seconds at 95 °C and 30 seconds at the melting temperature of the specific primer pair. Triplicate experiments were performed to analyze the gene expression of Us5 and β-actin, and the relative transcription level of the DPV Us5 gene was calculated using the 2 −ΔCt method simplified from the 2 −ΔΔCt method 28 . The relative expression levels of caspase-3 and caspase-9 were determined using β-actin as an endogenous control with the comparative Ct (−2 ΔΔin ) method and a real-time thermal cycler (CFX96 Bio-Rad, Hercules, CA, USA) 52 . The primers were designed with PRIMER 5 software and are listed in Table 2.
Pharmacological inhibition tests. Pharmacological inhibition tests were performed to confirm the kinetics of DPV Us5 27,53 . Four bottles of DEFs were prepared. Three were inoculated with DPV; one bottle contained no drugs, one bottle contained 300 μg/mL GCV (a DNA polymerase synthesis inhibitor), and one bottle contained 100 μg/mL CHX (a protein synthesis inhibitor). The fourth bottle contained untreated mock-infected cells. The infected cells were harvested 24 h after infection and washed twice with PBS. Total RNA was extracted and cDNA was synthesized as described above for RT-PCR. The Us5 gene type was identified by PCR (Us2 was used as a late gene control) 29 . The primers were designed with PRIMER 5 software and are listed in Table 2.
Us5 expression analysis by Western blotting. Western blotting was conducted as described in a previous study 54 . DEFs transfected with the empty pCAGGS plasmid and the pCAGGS-Us5 plasmid were collected at 48 h, and the protein expression was confirmed by Western blotting. A duck anti-DPV polyclonal antibody (at a final dilution of 1:500) was used as the primary antibody, and a goat anti-duck IgG antibody (at a final dilution of 1:2000) was used as the secondary antibody. In addition, DPV-infected cells and noninfected cells were collected at 6 h, 12   www.nature.com/scientificreports www.nature.com/scientificreports/ rabbit anti-DPV-Us5 polyclonal antibody (at a final dilution of 1:1000) was used as the primary antibody, and a goat anti-rabbit IgG antibody (at a final dilution of 1:3000) was used as the secondary antibody. Subcellular localization of DPV Us5 by IFA. IFA was conducted using a standard procedure 27 . Briefly, pCAGGS-Us5 plasmid-transfected cells and pCAGGS plasmid-transfected cells were collected at 48 h, and DPV-infected cells and noninfected cells were collected at 6 h, 12 h, 24 h, 36 h, 48 h, 54 h, and 60 h. The cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed three times with PBST, permeabilized with 0.25% Triton X-100 in PBS for 30 min at 4 °C and blocked for 1 h with 5% BSA PBS at 37 °C. Additionally, the cells were incubated for 1 h with the rabbit anti-DPV-Us5 polyclonal antibody at a 1:400 dilution at 37 °C. After the cells were washed, a 1:1000 dilution of goat anti-rabbit IgG-FITC secondary antibody was prepared in 1% BSA PBS and incubated with the cells for 1 h at 37 °C. Then, the cell nuclei were stained with DAPI for 15 min at room temperature. Finally, The cytoskeleton were stained with Phalloidin for 30 min at room temperature. After the coverslips were sealed on glass slides with glycerin buffer, the cells were examined with a confocal microscope (Nikon A1, Japan). DAPI staining of nuclei. DAPI staining of nuclei was conducted using a standard procedure 27 . Briefly, H 2 O 2 -treated cells and non-H 2 O 2 -treated cells were collected at 12 h and fixed with 4% paraformaldehyde for 60 min. The solution was permeabilized for 30 min and stained with DAPI for 10 min. The nuclei were examined with a confocal microscope (Nikon A1, Japan).

Apoptosis of DEFs detected by TUNEL assay.
Apoptosis was detected using an in situ cell apoptosis detection kit (POD) (Boster Biological Technology, USA). Briefly, cells were fixed with 4% paraformaldehyde for 60 min and incubated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining reaction mixture containing the enzyme solution and label solution prepared according to the manufacturer's protocol. The chromogenic substrate 3,3′-diaminobenzidine (DAB) was then added to visualize the TUNEL staining. The apoptotic nuclei were yellow, and the apoptotic cells were observed under the microscope.
Caspase-3, caspase-7, and caspase-9 activity detection with a Caspase-Glo 3/7 assay kit and a Caspase-Glo 9 assay kit. The activity of caspase-3/7 and caspase-9 was measured using a Caspase-Glo 3/7 assay kit and a Caspase-Glo 9 assay kit, respectively (Promega, Madison, Wisconsin, USA), according to the manufacturer's instructions. Briefly, after the treated adherent cells were removed, the cells were collected into EP tubes. The cells were counted with a blood cell counting plate. From each sample, approximately 10,000 cells were added to 100 µL of Caspase-Glo reagent in a 96-well white-wall plate and incubated for 30 min. Luciferase activity was detected with a multifunctional microplate reader (Thermo Scientific, Massachusetts, USA).

Mass spectrometry.
Purified virion samples were separated by 12% SDS-PAGE. The gel was stained with Coomassie brilliant blue (Sigma-Aldrich) and sent to Sangon Biotech Company (Shanghai, China) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis 27 . The details of LC-MS/MS, in-gel trypsin digestion, and the database searches were the same as those described in a previous study 55-57 . Flow cytometric analysis of apoptosis. The apoptotic cells were detected by flow cytometry (FCM); 5 µL of Annexin V-Fluorescein isothiocyanate (V-FITC) (BD Pharmingen, USA, 51-65874X) and 5 µL of propidium iodide (PI) (BD Pharmingen, USA, 51-66211E) were added to 100 µL of cell suspension and were incubated at 25 °C in the dark for 15 minutes. Annexin binding buffer (450 µL) (BD Pharmingen, USA, 51-66121E) was added to the mixture, and the percentage of apoptotic cells was assayed by FCM within 1 hour. Cell cycle was detected by FCM.
Statistical analysis. The data are expressed as the means ± S.D. Statistical analysis was performed with Student's t-test (GraphPad Prism 6); *p < 0.05 and **p < 0.01 indicate statistical significance compared with the control.