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Avibirnavirus VP4 Protein Is a Phosphoprotein and Partially Contributes to the Cleavage of Intermediate Precursor VP4-VP3 Polyprotein

Birnavirus-encoded viral protein 4 (VP4) utilizes a Ser/Lys catalytic dyad mechanism to process polyprotein. Here three phosphorylated amino acid residues Ser538, Tyr611 and Thr674 within the VP4 protein of the infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, were identified by mass spectrometry. Anti-VP4 monoclonal antibodies finely mapping to phosphorylated (p)Ser538 and the epitope motif 530PVVDGIL536 were generated and verified. Proteomic analysis showed that in IBDV-infected cells the VP4 was distributed mainly in the cytoskeletal fraction and existed with different isoelectric points and several phosphorylation modifications. Phosphorylation of VP4 did not influence the aggregation of VP4 molecules. The proteolytic activity analysis verified that the pTyr611 and pThr674 sites within VP4 are involved in the cleavage of viral intermediate precursor VP4-VP3. This study demonstrates that IBDV-encoded VP4 protein is a unique phosphoprotein and that phosphorylation of Tyr611 and Thr674 of VP4 affects its serine-protease activity.


Introduction
Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius and causes severe immunosuppression and mortality in young chickens. The IBDV genome is characterized by a bisegmented double-stranded RNA (segments A and B). The smaller segment B only encodes the VP1 with a molecular weight of 90 kDa. VP1 is the putative RNA-dependent RNA polymerase which interacts with the viral genome [1,2] and is involved in IBDV mRNA translation via association with the carboxy-terminal domain of the eukaryotic translation initiation factor 4AII [3]. It has also been demonstrated to affect viral replication kinetics and modulate the virulence [4][5][6]. The larger segment A contains two partially overlapping open reading frames (ORFs) [7]. The smaller ORF encodes the VP5 protein, a 17-kDa nonstructural protein which interacts with host proteins, subunit p85α of PI3K and voltage-dependent anion channel 2, and plays important roles in regulating virus release and apoptosis [8][9][10]. The larger ORF encodes a 110-kDa polyprotein precursor that can be cleaved by the proteolytic activity of VP4 into the precursor of VP2 (pVP2, 48 kDa), VP3 (32 kDa) and VP4 (28 kDa) [11]. During virion maturation, pVP2 is further processed into the mature capsid protein VP2 (41 kDa) and four small peptides [12][13][14]. VP2 carries the major immunogenic determinants [15,16] and contributes significantly to apoptosis, cell tropism, virulence and pathogenicity of virulent IBDV [17][18][19]. VP3, a major immunogenic and scaffolding protein of IBDV [20,21], was found to interact with VP1 [22] and bind to the viral dsRNA forming ribonucleoprotein complexes [23], as well as thought to be a key organizer in virion morphogenesis [21]. VP4, as the viral protease of Birnaviruses, has been proposed to utilize a Ser/Lys catalytic dyad mechanism to process the polyprotein [11,24]. VP4 forms regular needle-like structures called type II tubules within the cytoplasm and nucleus of IBDV-infected cells [25]. Meanwhile, current research data shows that E.coli-expressed VP4 protein can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni 2+ ions [26].
Recently, more attention has been paid to the functions of viral protein phosphorylation during virus infection. Phosphorylation at the Ser224 site of ICP0 of herpes simplex virus type 1 is known to be required for efficient viral replication [27]. Phosphorylated sites at Ser479 and Ser510 of the N protein in measles virus are important for the activation of viral mRNA transcription and/or replication of the genome in vivo [28]. In hepatitis C virus, the phosphorylated site at Ser222 of NS5A functions as a negative regulator of RNA replication [29]. The phosphorylation of Ser60, Ser64, and Thr62 of the P protein of vesicular stomatitis virus is critical for viral genome RNA encapsidation and template function [30]. Dephosphorylation of VP40 at sites Tyr7, Tyr10, Tyr13 and Tyr19 of Marburg virus impairs its ability to recruit nucleocapsid structures into filopodia, causing release of virions with low infectivity [31]. Phosphorylation of the capsid protein of West Nile virus mediated by protein kinase C has been shown to enhance its binding to HDM2 protein and importin and subsequently induce p53-dependent apoptosis [32]. The protein kinase A-mediated phosphorylation of Vpr at Ser79 site was found to be crucial for cell cycle arrest in HIV infection [33]. All these examples illustrate that phosphorylation of viral proteins plays important roles in regulating processes such as gene expression, viral replication and cell cycle arrest during viral infection.
Other than being reported as a serine-protease and an intracellular tubule type II, the VP4 protein of IBDV was also found to have novel roles as a biomarker for discriminating between pathogenic and nonpathogenic IBDV infections [34] and an inhibitor suppressing the expression of type I interferon via interaction with the glucocorticoid-induced leucine zipper [35]. In our previous proteomic analysis of IBDV-infected cells, different protein spots of VP4 were evident in the two-dimensional electrophoresis (2-DE) gel [36]. As it was of interest to learn whether these spots represented post-translational modifications, in this study we identified the phosphorylation sites of VP4 and generated monoclonal antibodies (mAbs) against phospho-and nonphospho-VP4 protein. Additionally, an in-depth analysis of the protease activity of phospho-VP4 was conducted.
Generation and fine mapping epitopes of mAbs against IBDV VP4 protein IBDV-infected cell lysates were separated by 12% SDS-PAGE, and the VP4 specific protein band was purified by eluting the protein from the excised gel in a dialyzer (Serva, Heidelberg, Germany) by electrophoresis in protein electrophoresis buffer (25 mM Tris base, 192 mM glycine, 3.5 mM SDS). The purified VP4 preparation was used as an immunogen and injected intraperitoneally into SPF BALB/c mice in order to generate mAbs to VP4 of IBDV as described previously [40,41]. Reactivities of anti-VP4 mAbs were screened by an indirect immunofluorescence assay (IFA) and Western blot analysis. The phosphorylated and dephosphorylated antigenic epitope peptides of IBDV-VP4 (Table 1) were designed using three on-line prediction software programs (http://www.cbs.dtu.dk/services/BepiPred/, www. epitope-informatics.com/Links.htm, http://www.imtech.res.in/raghava/cbtope/submit.php) and synthesized using a Symphony Multiplex Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ). Peptide ELISA and peptide dot-ELISA were performed to test the reactivities of the mAbs with peptides as described previously [40]. After an immunoactive peptide was identified, its N-truncated, C-truncated and Ala-substituted derivatives were further synthesized and used to define the epitope motif by ELISA.
The letter "c" means cysteine appended to the Sulfo-SMCC cross-linker. Mutated residues are underlined.

Immunofluorescence staining
The indirect immunofluorescence assay was performed as described previously [36]. DF-1 cells or 293T cells were seeded in 96-well plates (Corning, New York, NY) or 35-mm glass bottom dishes (Shengyou Biotechnology, China), infected with IBDV or transfected with wild-type A segment, with wild-type VP4 or Ala/Asp VP4 mutants. The mouse mAb to phospho-VP4 or nonphospho-VP4 was used as primary antibody. The direct immunofluorescence assay was performed with Alexa Fluor 555 or Alexa Fluor 488 labeled mAbs to phospho-VP4 and nonphospho-VP4 on IBDV-infected DF-1 cells. Mock cells were used as negative controls. The nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). The stained cells were washed three times with PBST and subsequently examined under a Zeiss LSM510 laser confocal microscope.

Site-directed mutagenesis and in vivo transfection
Various plasmids were generated using the pCI-neo-VP4 or pEGFP-VP4 plasmid as a template and PCR primers shown in Table 2 for site-directed mutagenesis. The PCR for dephospho-or phospho-mimicking recombinant VP4 was performed in a final volume of 25 μl containing 2.5 μl 10× Pyrobest buffer II (5 U/μl), 0.5 μl dNTP mixture (10 mM), 1 μl primers, 15 ng DNA template and 0.25 μl Pyrobest DNA polymerase, with the following conditions: denaturation at 94°C for 4 min, followed by 20 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 6 min, with a final elongation step at 72°C for 10 min. The PCR products were digested with Dpn Ι at 37°C for 1 h and confirmed by enzyme digestion and DNA sequencing. DF-1 cells were transfected with these recombinant plasmids using Lipofectamine 2000. At 6 and 15 h post-transfection, the cells were observed directly under the fluorescent microscope (pEGFP-transfected) or immunostained with anti-VP4 mAbs followed by FITC-conjugated secondary antibodies (pCI-neo-transfected). In vivo and in vitro proteolytic activity assay Dephospho-or phospho-mimicking segment A with or without single and multiple mutations within VP4 were generated using pCI-neo-TNT-A as a template (unpublished data) following the same procedure as mentioned above. For the in vivo assessment of protease activity, 293T cells were transfected with various purified recombinant plasmids individually using Lipofectamine 2000 for 24 h. The cells were rinsed with PBS and lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% TX-100, 0.5% sodium deoxycholate, 50 mM NaF and 0.2 mM Na 3 VO 4 ). The supernatant was subjected to 12% SDS-PAGE and Western blot analysis to assess the cleavage activity of mutant segment A using the VP3 and nonphospho-VP4 (P530) mAbs. The purified recombinant plasmids were further used to test the expression of polyprotein VP2/4/3 using the TNT T7 Quick Coupled Transcription/Translation System according to the manufacturer's instructions. Briefly, 40 μl T7 Quick master mix, 1 μl methionine (1 mM), 7 μl nuclease-free water and 2 μl plasmid (500 ng/μl) were incubated at 30°C for 90 min. The resultant samples were subjected to 12% SDS-PAGE and Western blot analysis to assess the cleavage activity of mutant segment A using the anti-VP3 and nonphospho-VP4 (P530) mAbs.

Co-immunoprecipitation (Co-IP) assay
Co-IP experiments were performed as previous described [39]. Briefly, IBDV-infected and mock-infected cells were lysed with 500 μl NP-40 lysis buffer at 4°C for 30 min. Cell lysates were clarified by centrifugation at 8,000 × g for 10 min, and the supernatants were diluted with 500 μl PBS. The anti-phospho-VP4 mAb or anti-P530-VP4 mAb was added to the supernatants and incubated at 4°C for 8 h, and protein-A/G plus beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the mixtures and incubated at 4°C for 8 h. Subsequently, the beads were washed with PBS three times and boiled with loading buffer, and the supernatants were prepared for SDS-PAGE and Western blot analysis.

MS/MS identification of phosphorylated amino acid residues within VP4 protein
To identify whether the VP4 protein of IBDV is phosphorylated, IBDV-infected DF-1 cells were treated with 2-DE lysis buffer and separated by SDS-PAGE. Protein bands were excised manually from gels and sequentially subjected to in-gel digestion and MS identification by LC-MS/MS. As shown in Table 3, three phosphorylated peptides with the putative phosphorylation sites at Ser538 (S538), Tyr611 (Y611) and Thr674 (T674) were identified in VP4 protein of IBDV, indicating that the IBDV-encoded VP4 protein is a multi-site phosphorylated protein.

Generation and specificity of mAbs to VP4 protein of IBDV
The VP4 protein generated in IBDV-infected DF-1 cells was confirmed by Western blot analysis using the anti-VP4 pAb (S1 Fig), and then the specific protein band was gel-purified after separation by 12% SDS-PAGE. To prepare the mAb recognizing IBDV VP4, BALB/c mice were immunized with VP4 as the antigen. Ultimately, five hybridoma cell lines (4A8, 5B2, 5C7, 7A4 and 7H8) secreting mAbs to the VP4 of IBDV were cloned. Western blot analysis showed that these generated mAbs could specifically react with VP4 protein expressed in both VP4-transfected and IBDV-infected DF-1 cells (Fig 1A). IFA also indicated that these mAbs could recognize the VP4 protein in IBDV-infected cells (Fig 1B). However, by Western blot and IFA, these mAbs did not react with the viral proteins VP1,VP2, VP3 and VP5 of IBDV Note: The bold rows revealed the identified peptides that contained a phosphorylated amino acid.
"#" is the phosphorylated amino acid residues.

Fine-mapping of linear antigenic epitope on VP4 protein of IBDV
Based on the LC-MS/MS identification data, the antigenic epitopes recognized by anti-VP4 mAbs were finely analyzed with a series of overlapping and phosphorylated linear peptides synthesized by the PEPSCAN technique (Table 1). Of the five mAbs tested in the peptide ELISA and peptide dot-ELISA, the mAbs 7A4 and 7H8 could react with the unphosphorylated peptide Pep515-558, while none of the synthesized peptides reacted with the mAbs 4A8, 5B2 and 5C7 (Fig 2A). Subsequently, in N-and C-terminally truncated peptides derived from unphosphorylated Pep515-558, the mAbs 7A4 and 7H8 could still react with the unphosphorylated peptide Pep530-536 (Fig 2B), indicating that the anti-VP4 mAbs 7A4 and 7H8 recognized the same linear B-cell epitope with the amino acid motif 530 PVVDGIL 536 (P530). The substitution analysis further revealed that the residues 531 VV 532 were dispensable for the antigenicity of the epitope, but any change of the residues of 530 P and 533 DGIL 536 resulted in the loss of reactivity of the mAb (Fig 2B). These results indicated that 530 P and 533 DGIL 536 are the crucial residues of the P530 epitope (P530 mAb). Interestingly, as shown in Fig 2C,

Phosphorylated VP4 exists in complexes with different isoelectric points
To analyze the intracellular VP4 with and without the Ser538 phosphorylation, IBDV-infected DF-1 cells were separated into cell membrane, cytosol, nuclear and cytoskeletal fractions. As shown in the Western blot analysis of IBDV-infected cells (Fig 3A), the VP4 protein recognized   . Meanwhile, two of five protein spots could be recognized with the pSer538 mAb ( Fig 3B Lower panel), and the pSer538-VP4 protein represented a lower proportion than that of the P530-VP4 protein. Additionally, Co-IP analysis demonstrated that the pSer538 mAb-recognized VP4 molecule also could be bound by the P530 mAb (Fig 3C), while the pSer538 mAb-recognized VP4 protein co-localized with the P530 mAb-reacted VP4 protein (Fig 3D). These results indicated that VP4 contains both the pSer538 and P530 epitopes ( Fig  3C and 3D). Generally, the results demonstrated that IBDV VP4 was mainly detected within the insoluble cytoskeletal fraction, and pSer538-VP4 protein exists in complexes with different isoelectric points and is a minor protein in comparison with the P530-VP4.

Phosphorylation modification is unrelated to intracellular accumulation of VP4
Substitution of phosphorylated sites with Asp (D) or Glu (E) was commonly used to mimick the phosphorylation site and study its functions [27,29,30]. To further analyze whether the phosphorylation modification regulates subcellular distribution, the sites Ser538, Tyr611 and Thr674 within VP4 of IBDV were mutated into Ala or Asp, and a series of dephosphorylated VP4 mutants were constructed. In cells transfected with the pEGFP-VP4 mutants (Fig 4) or pCI-VP4 mutants (S2 Fig), the expressed VP4 proteins that were mutated to Ala or Asp at the sites Ser538, Tyr611 and Thr674, aggregated into the mass-like structure of wild-type VP4 but not the rod-like or needle-like or filamentous structure VP4 in IBDV infected cells (Fig 3D). However, the VP4 aggregation is not easy to be observed at 6 h post-transfection in the VP4 mutants S538/T674A-, Y611/T674A-and S538/Y611/T674A-dephosphorylated cells (Fig 4), suggesting that the dephosphorylation of these sites potentially postpones the VP4 expression. Thus, the dephosphorylation of Ser538, Tyr611 and Thr674 did not influence the aggregation of VP4 protein, and therefore the phosphorylation of these sites may not involve the VP4 aggregation.

The phosphorylated Tyr611 and Thr674 within VP4 protein is involved in cleavage of intermediate precursor VP4-VP3
The VP4 protein of IBDV is a protease that plays an important role in the maturation of viral protein precursor. To detect whether the phosphorylation modification is involved in the  proteolytic activity, various mutants of the segment A with the dephosphorylated and mimicked sites of Ser538, Tyr611 and Thr674 were constructed and transfected into cells for Western blot analysis. Only T674A/D and Y611D substitutions partially abolished the polyprotein cleavage, and the intermediate precursor VP4-VP3 protein band with a molecular weight of approximately 60 kDa was detected both with the anti-VP3 and anti-VP4 mAbs; meanwhile, substitutions at sites S538A/D and Y611A did not affect the VP4 and VP3 protein maturation (Fig 5A). Similar proteolytic activity in vitro was also analyzed in TNT tests. As shown in Fig  5B, any substitution at site T674 within VP4 decreased its proteolytic activity, and the intermediate precursor VP4-VP3 protein was detected. The single Ala substitution at S538 and Y611 did not affect the function of VP4 protein, while the Asp substitution of the site Y611 which mimicked Tyr phosphorylation partially affected the proteolytic activity. In further co-localization analysis (Fig 5C and S3 Fig), Table 4.

Discussion
In this report, phosphorylation of the IBDV VP4 protein at the amino acid residues Ser538, Tyr611 and Thr674 were identified by LC-MS/MS spectrum analysis (Table 3), demonstrating that the virally encoded VP4 protein is a phosphoprotein. The commercial mAbs against pSer (PSR-45, Sigma), pThr (PTR-8, Sigma) and pTyr (PT-66, Sigma) are widely used to investigate phosphorylation modifications. However, in our study these commercial antibodies failed to specifically detect the phosphorylation of Ser538, Tyr611 and Thr674 within the IBDV-encoded VP4 molecule (S4 Fig), indicating the need of developing new antibodies to detect the phosphorylated VP4. Therefore, a mAb specific for the pSer538 of the VP4 (Figs 1 and 2) was generated in this study, providing an important tool for analyzing the novel function of the phosphoprotein VP4. The phosphorylation of viral proteins has not been reported for all members of the family Birnaviridae. In the present study, the Western blot results showed that the VP4 is abundant in all cell fractions, including the nucleus, cytoplasm, membrane and insoluble cytoskeleton, and pSer538-VP4 accounted for a small proportion of the insoluble cytoskeletal fraction. Correspondingly, in the 2-DE blots, the abundant VP4 protein and a small amount of pSer538-VP4 protein were detected in the same protein spot (Fig 3B Middle panel), as well as in two different protein spots (Fig 3B Lower panel). Further immunofluorescence analysis revealed co-localization of the VP4 proteins recognized by the pSer538 and P530 mAbs (Fig 3D). Similarly, previously published proteomic data have shown different 2-DE protein spots representing the viral VP4 protein in IBDV-infected CEF cells and bursal lymphocytes [36,38]. These results demonstrated that the VP4 in IBDV-infected cells with pSer538 modification was a minor and site-directed mutation of Ala or Asp substitution with the vector pEGFP-C2 and transfected into DF-1 cells. Subcellular distribution of each mutant was observed with a laser Zeiss LSM510 laser confocal microscope. Different time points post-transfection are labeled. Nuclei were counterstained with DAPI. doi:10.1371/journal.pone.0128828.g004 The Phosphorylation of Infectious Bursal Disease Virus VP4 Granzow et al. [25] reported that the intracellular type II tubule contains the IBDV VP4 protein. However, our unpublished co-localization experiments data shows there is no overlap between the cytoskeleton and mass-like, filamentous structure VP4 protein, furthermore, continuous accumulations of VP4 structures occupy a large amount of intracellular space and result in the mechanical destruction of host cytoskeletal elements (data not shown). Since collapse of the host cytoskeleton typically affects cellular integrity, this will lead to cell lysis facilitating virion egress at later stages of infection. Whether type II tubules are involved in VP4 phosphorylation is currently unknown. In the present study, the single or combined mutants mimicking dephosphorylation and phosphorylation at Ser538, Tyr611 and Thr674 of VP4, still formed a mass-like structure (Fig 4), but not a rod-like or needle-like or filamentous structure of wild-type VP4 in IBDV-infected cells (Fig 1B), indicating that these three identified phosphorylated sites within VP4 are unrelated to the formation of intracellular type II tubules. Antibody response to the IBDV VP4 protein has been reported as a biomarker discriminating the pathogenic and nonpathogenic IBDV infection [34], and VP4 protein has been reported to be an inducer of suppressing type I interferon expression via interaction with the glucocorticoidinduced leucine zipper [35]. Whether the roles are relevant to the phosphorylation of VP4 requires more in-depth investigation.
In the present study, the subcellular fractionation analysis demonstrated an abundant amount of VP4 protein in the cytoskeleton fraction ( Fig 3A). However, whether there is an association between the cytoskeleton and VP4 protein is not known. The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during movement depends on the cytoskeleton, which consists of actin filaments, microtubules and intermediate filaments, an interconnected network of filamentous polymers and regulatory proteins [42]. Many proteins interact with actin through one of the following actin-binding motifs: calponin homology domain [43], ADF-H domain [44], gelsolin homology domain [45] or thymosin β4/WH2 (WASP homology domain-2) domain, a~35 residue actin monomer-binding motif [46]. The critical and conserved actin-binding residues are Ile, Leu and Arg/Lys all in WH2 domains [47]. Based on a publicly available service, http://elm.eu.org/, we found a potential WH2 motif within the VP4 protein of IBDV, which contains conserved marker residues 535 Ile, 542 Leu and 543 Arg. Thus, it may not be surprising that the VP4 protein mainly resides in the cytoskeleton fraction (Fig 3A), and this observation may be suggestive of a potential relationship between VP4 protein and actin that is worthy of further study.
pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephosphoand phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins. doi:10.1371/journal.pone.0128828.g005 The Phosphorylation of Infectious Bursal Disease Virus VP4 Table 4. The summary of proteolytic cleavage result affected by a series of substitution in this study. Site-directed mutagenesis studies on the VP4 protease have shown that the conserved catalytic residues serine 652 and lysine 692 in IBDV are essential for polyprotein processing [11,48]. Similar results were also found in the VP4 protease of infectious pancreatic necrosis virus (IPNV, serine 633 and lysine 674) [49] and the blotched snakehead virus (BSNV, serine 692 and lysine 729) [50] and Tellina virus-1 (TV-1, serine 738 and lysine 777) [51]. The VP4 protease of the Birnaviridae family therefore is proposed to utilize a serine/lysine catalytic dyad mechanism to catalyze the processing of the polyprotein [11,48,49,[51][52][53]. The replacement of serine by lysine in the AXAAS motif in the VPX-VP4 boundary ( 485 AQAASGTARAASG KARAAS 504 ) has been found to influence polyprotein processing by VP4. Furthermore, mutation of 514 D ( 510 TLAADK 515 ) was shown to prevent cleavage at the VPX-VP4 junction, while the H547P mutation abolished the polyprotein processing completely, indicating that this histidine plays a very important role in the VP4 protease catalytic activity [54]. Furthermore, the atomic structures of birnavirus VP4 proteases reveal that O γ1 of T712, T655 and T760 (T674 in IBDV) in the polyprotein243 of BSNV, IPNV and TV-1, is a critical donor for generating the deacylating (catalytic) water interacting with N 03B6 of the lysine general base (Lys 729, Lys 674 and Lys 777 corresponding to BSNV, IPNV, TV-1 respectively) to form the catalytic activity domain, and the residue T674 but not Y611 in IBDV is conserved within the Birnaviridae family [51][52][53]. In this study, Ala or Asp substitutions (dephospho-or phospho-mimicking mutations) at the pThr674 site in vivo and in vitro both led to a marked decrease of proteolytic enzyme activity, estimating that the mutation of Thr674 abolish the ability for the lysine to function as a general base and inhibit polyprotein processing. Substitution of Asp but not Ala at pTyr611 also led to minor negative effects on proteolytic activity. Ser538 is not the active site of serine protease, therefore, Ala or Asp substitution at the pSer538 site does not affect proteolytic enzyme activity (Fig 5A and 5B). Our results above show that Tyr611 and Thr674 phosphorylation affected the maturation of intermediate precursor VP4-VP3, and further in-depth investigation about pVP2-VP4 cleavage will be followed up. In all, our results reveal that pTyr611 and pThr674 may play a partial role in the process of polyprotein cleavage.