Characterization of the duck enteritis virus UL55 protein
- Ying Wu1,
- Anchun Cheng1, 2, 3Email author,
- Mingshu Wang1, 2, 3Email author,
- Shunchuan Zhang†1,
- Dekang Zhu1, 2,
- Renyong Jia1, 2, 3,
- Qihui Luo3,
- Zhengli Chen3 and
- Xiaoyue Chen1, 2
© Wu et al; licensee BioMed Central Ltd. 2011
Received: 9 March 2011
Accepted: 24 May 2011
Published: 24 May 2011
Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function.
The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells.
In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.
Duck enteritis virus (DEV), alternatively known as Duck plague virus (DPV), is a fatal pathogen of the family Anatidae of the order anseriformes, leading to an acute, febrile, contagious, and septic disease to waterfowls of all ages. The resulting disease designated as duck virus enteritis (DVE) has caused serious losses in commercial duck production in domestic and wild waterfowl since it was firstly discovered in Netherlands. To our knowledge, DEV has been clustered to the subfamily of alphaherpesvirinae according to the report of the Eighth International Committee on Taxonomy of Viruses (ICTV). However, it has not been classified to any genus yet.
The genome of DEV is composed of a linear, double stranded DNA. In recent years, due to the advent of molecular biology and advancements in research related to it, a lot of DEV genes has been identified, such as US2 to US5 and US10 genes[4, 5], UL6 and UL7 genes, UL10, UL24, TK and gH genes, UL27, UL31, UL35, UL38, UL45 to UL47 [13–15], UL51, gK, gC, gE, gI and so on. Even so, a great deal of unkown DEV genes remain to be clarified to facilitate the investigation of this virus. DEV UL55 gene [GenBank:EU071034] was a kind of that gene whose properties and functions has not been elucidated yet since it was identified in our laboratory in 2006. To our best knowledge, the UL55 gene of alphaherpesviridae was supposed to be a late gene. Reports about HSV-2 UL55 protein revealed that the product of HSV-2 UL55 gene may play an accessory role in virion assembly or maturation, but the corresponding homologue gene of EHV-1 was supposed to mediate persistent infection. However, the characterization of the DEV UL55 protein (pUL55) remains unclear.
To investigate the characteristics of DEV pUL55, we amplified the DEV UL55 gene by PCR and constructed a recombinant plamid pET32a(+)/UL55 for prokaryotic expression. The expression of recombinant pUL55 was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) and supposed to be maximum after optimization. Polyclonal antibody was prepared by immune rabbits with purified pUL55, and then detected by agar immunodiffusion and viral neutralization test. It was subsequently used to determine the expression and subcellular localization of pUL55 in DEV infected cells. This work was supposed to facilitate the understanding of DEV pUL55 and its functional location in infected cells.
Computer analysis of DEV pUL55
A complete ORF of DEV CHv strain was first identified in our laboratory and designated as UL55 gene. It was about 561 bp and expected to encode a protein comprising 186 amino acids with a putative molecular mass of 20.7981 KDa. A series of bioinformatics aided tools were used to analyze the intracelluar location of pUL55 : PSORT II Prediction(from the website http://psort.nibb.ac.jp/form2.html), TargetP 1.1 (from the website http://www.cbs.dtu.dk/services/TargetP/), SignalP 3.0(from the website http://www.cbs.dtu.dk/services/SignalP), TMHMM 2.0 server (from the website http://www.cbs.dtu.dk/services/), PredictNLS server(from the website http://www.rostlab.org/services/predictNLS/), CSS-Palm 2.0 online server (from the website http://csspalm.biocuckoo.org/online.php), and the Golgi predictor (from the website http://ccb.imb.uq.edu.au/golgi/golgi_predictor.shtml). Prediction of them were based on the putative amino acid sequence of pUL55.
Cells, viruses, serums, and vectors
Duck embryo fibroblasts (DEF) were cultured in modified eagle's medium (MEM)(Gibco-BRL) supplemented with 10% fetal bovine serum (FBS)(Gibco-BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. MEM medium supplemented with 2-3% FBS was used for virus infection. DEV CHv strain and rabbit anti-DEV serum were obtained from Key Laboratory of Animal Disease and Human Health of Sichuan Province[32, 33]. Besides, Escherichia coli strain DH5α, Escherichia coli BL21 (DE3) and expression vector pET-32a(+) were preserved in our laboratory.
Expression and purification of recombinant UL55 protein
The amplified DEV UL55 gene was directionally cloned to pMD18T as previously discribed. After confirmation by sequencing, the digested gene fragment of the recombinant plasmid pMD18-T/UL55 (retrieving by TIANgel Midi purification Kit) was directionally ligated into the previously BamH I/Xho I- digested expression vector pET32a(+), gernerating a recombinant plasmid pET32a(+)/UL55. Subsequently, the PCR, restriction enzyme digestion and DNA sequencing (TaKaRa) tests were performed to ensure the correct insertion. After that, the positive recombinant plasmids were transformed to Escherichia coli BL21 (DE3) for expression by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The tempreture and duration of IPTG and its working concentration were optimized as descried to maximize the expression of pUL55. Cells were centrifugated and lysed in 5×sample buffer (0.1 M Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 0.1 M DTT, pH 6.8), then analyzed by SDS-PAGE. The uninduced control culture and the vector control culture were analyzed in parallel.
The recombinant pUL55 was purified under denaturing condition by repeated washing. The induced cells were centrifugated at 10,000 rpm/min for 10 min, and resuspended in 20 mM Tris buffer (pH 8.0) with the addition of 0.1 mg/ml lysozyme (0.1 mg/ml) at -20°C overnight. The cell lysate was then sonicated on ice for 5 min at an amplitude of 30% with a 30 s pulse frequency. After 10 min centrifugation at 10,000 rpm/min, the supernatant (soluble fraction) and pellets (insoluble fraction) of it were collected respectively for SDS-PAGE analysis. Result demonstrated that the recombinant pUL55 has formed inclusion bodies (IB). The pellets were resuspended in 20 ml washing buffer (2 M urea, 50 mM Tris-HCl buffer, 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100, pH 8.0) under constant stirring for 10 min, then followed by centrifugation at 10,000 rpm/min for 10 min at 4°C. The above steps were repeated five times to release the trapped protein. The suspension was finally centrifuged at 10,000 rpm/min for 10 min at 4°C, and resuspended in denaturing buffer containing 8 M urea, 10 mM PBS, 50 mM Tris-HCl, 50 mM NaCl, 10% glycerine, pH 8.0. The purity of pUL55 was tested by SDS-PAGE.
Western blotting assays
Western blotting assay was performed using the purified rabbit anti-DEV (diluted 1:200) IgG to characterize the reactivity and specificity of the recombinant pUL55. The purified recombinant pUL55 were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane at 120 V for 1.5 h in a BioRad mini Trans-Blot electrophoretic transfer cell (BioRad, Shanghai, China). Blocking the membrane with 10% skimmed milk in TBST (Tris-buffered saline with 0.1% Tween-20, pH 8.0) for 1 h at 37°C or overnight at 4°C. Sequently, the membrane was incubated with appropriate dilution of rabbit anti-DEV (diluted 1:200) serum for 1 h at 4°C overnight. After washing three times, the HRP-conjugated goat anti-rabbit IgG (diluted 1:3000) was added for incubation. Pre-serum came from non-immune healthy rabbit blood was disposed parallelly for control. One hour later, washing the membrane with TBST as before, followed by 3 min for color development with substrate solution (DAB 3'3'-Diaminobenzidine tetrahydrochloride peroxidase) at 37°C. The reaction was terminated by thoroughly washing with distilled water.
Preparation of polyclonal antibody against recombinant pUL55
Renaturation of recombinant pUL55 was carreied out by dilution method and gradient dialysis. Firstly, the refolding buffer (1 mM EDTA, 0.15 M NaCl, 50 mM Tris-HCl, 1 mM GSSG, 1 mM GSH and 1% Arginine, pH 8.0) was added to the denatured pUL55 slowly until the urea concentration reached 6 M. Sequently, the partly refolded protein was dialyzed in different concentrations of urea buffer solution (6 M, 4 M, 3 M, 2 M) containing 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA and 10% glycerine, pH 8.0 at 4°C. Changing the dialyzate of each at least three times a day. At last, the aggregation was removed by centrifugation and the supernatant was collected as soluble refolded protein.
For preparation of polyclonal antibodies, male New Zealand white rabbits were first immunized intradermally with a mixture of 0.5 mg renatured recombinant pUL55 and an equal amount of complete Freund's adjuvant (Sigma, Shanghai, China). Two weeks later, 0.75 mg purified fusion pUL55 and an equal amount of Freund's incomplete adjuvant were used for secondary immunity. After that, the rabbits were boosted subcutaneously with 1.0 mg each of recombinant pUL55 and an equal amount of incomplete Freund's adjuvant at a 1-week interval. Seven days later, the rabbits were injected intravenously with 0.1 mg purified pUL55 each. At last, serums were collected 17 days later. Control pre-immune serum was obtained from the non-vaccinated healthy rabbits.
The obtained rabbit polyclonal anti-serum against pUL55 was subsequently purified by ammonium sulfate precipitation and High-Q anion-exchange chromatography following the manufacturer's instructions. The purified IgG fraction was analyzed by 12% SDS-PAGE.
Agar diffusion reaction
Agar diffusion reaction was used to detect the reactivity and specificity of the purified UL55 anti-serum. One gram of agar was dissolved in 100 ml normal saline for the test. It was heated, cooled down to 55°C, and then poured into the plates to a thickness of 2 mm. After subsequent solidification with cooling, the agar was perforated with 3 mm diameter holes that may hold approximately 100 μl of solution. Twenty microliters each of the pre-immune serum, 1:2, 1:4, 1:8, 1:16 and 1:32 diluted anti-serum was added into the peripheral apertures. At last, 20 μl purified pUL55 was added into the central aperture. The plate was incubated at 37°C for 24 h before observation.
Viral neutralization test
Viral neutralization test was used to determine the neutralizing viral antibody titer of the obtained anti-serum. DEFs were prepared as we described above, and 350 μl of cell suspension was added to each well of the 48-well plate for incubation. Sequently, inactivated anti-pUL55 serums (56°C for 30 min) were serially diluted twofold from 1:1 to 1:32. Mixing 25 μl of the 200 TCID50 (TCID50 = 10-6.334) virus which was diluted from the virus stock suspension previously with an equal volume of serum dilution, and incubating it at 37°C for 1 h. When the cells grew into a monolayer, 50 μl of each incubated antiserum was inoculated onto the cells for infection. Meanwhile, seven contrast controls were set up for later observation: blank control 1:2, diluted anti-serum, 200 TCID50, 100 TCID50, 10 TCID50, 1 TCID50 and 0.1 TCID50 was respectively added to the cell culture. Each dilution of these invovled serums and viruses were tested in triplicate. After 1 h adsorption at 37°C, the cells were overlaid with the MEM maintenance media for incubation. Observation the cytopathic effect (CPE) of them timely.
The dynamic expression of UL55 protein in DEV-infected cells
DEFs infected and mock infected with DEV were harvested at 8 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h post-infection to determine the kinetics of pUL55 expression. Cells lysate were mixed with 5×SDS sample buffer and heated at 100°C for 10 min. Then centrifugalization it before SDS-PAGE. After gel separation, proteins were transformed onto PVDF membrane for western blotting. It's worth noting that, here purified DEV UL55 IgG (diluted 1:64) substitued DEV IgG for dynamic expession analysis.
Intracellular localization of UL55 protein in DEV-infected cells
Indrect immunofluorescent microscopy was used to investigate the intracellular location of pUL55 in infected cells. Preparing monolayers of DEFs as a matter of routine, cells were expected to grow on coverslips in six-well plates and they were supposed to be mock infected or infected with DEV. At different times (5.5 h, 11 h, 16 h, 22.5 h, 30 h, 35 h, 40 h, 45.5 h, 49 h, 54 h, 60 h, 70 h and 74 h post infection), cells were harvested and fixed with 4% paraformaldehyde overnight at 4°C. Sequently, they were washed with PBS buffer and permeabilized with 0.1% Triton X-100 for 30 min. After that, washing the cells with PBS contaning 0.1% tween-20 for three times before they were blocked with PBS containing 4% BSA for at least 1 h at 37°C. Then, the cells were incubated overnight with purified UL55 IgG (1:64 diluted) in PBS containing 1% BSA at 4°C. Three times washing had been performed as decribed above before they were treated with 1:100 diluted FITC conjugated goat anti-rabbit IgG (Sino American Biotechnology Co.) at 37°C for 1 h. The cell nuclei were visualized by 4', 6-diamidino-2-phenylindole (DAPI) counter-staining (5.0 μg/ml, Beyotime Institute of Biotechnology, Shanghai, China) after washing three times. The images were captured with fluorescence microscopy (Nikon, Japan).
Prediction of subcellular localization of DEV pUL55
Computer analysis of the subcellular localization of DEV pUL55 suggested that the pUL55 was mainly located in cytoplasmic (60.9%) of infected cells, then in cytoskeletal (17.4%), nuclear (13.0%), peroxisomal (4.3%) and mitochondria (4.3%) sequentially. However, according to the prediction, DEV pUL55 contained no potential mitochondrial targeting peptide, N-terminal signal peptides, transmembrane region and nuclear localization signal (NLS). Further, Golgi prediction results indicated pUL55 was not a Golgi type II membrane protein(Golgi localised transmembrane protein) since the index values of a Golgi protein should be geater than the threshold (20.005) while the index values of pUL55 was 0.
Expression and purification of UL55 recombinant protein
Verification the character of polyclonal antibody against DEV pUL55
Observation of the neutralization titer of the rabbit anti-pUL55 polyclonal antibody was detected by micro neutralization test. Calcutating 50% serum neutralized through Reed-muench method. As a result, the neutralization titer of the rabbit anti-UL55 polyclonal antibody was 1:7.484 (data not shown).
Dynamic expression of pUL55 in DEV-infected cells
Intracellular localization and distribution of DEV pUL55 in DEV-infected cells
The product of DEV UL55 gene which has been designated as pUL55, was a 186 amino acids protein encoded by a 561 bp ORF. In our research, a series of experiments were preformed to characterize the duck enteritis virus UL55 protein. As the first step towards studying the characterization of the DEV pUL55, the digested UL55 fragment was directionally inserted into the pMD18-T and pET32a(+) vector sequentially to constrcut recombinant plasmids (Figure 1). PCR, Restriction enzyme digestion and DNA sequencing were used to comfirm the correctness of insertion as described previously. The determined recombinant plasmid pET32a(+)/UL55 was transformed into Escherichia coli BL21 for prokaryotic expression. The optimal expression condition of recombinant pUL55 was induced by 0.2 mM IPTG at 37°C for 4 h. A 6×His-Tag fusion pUL55 approximately 40 KDa was collected as inclusion bodies in exprssion procedure and can be easily purified after washing five times under denaturing conditions. The refolded pUL55 could be recognized by rabbit anti-DEV IgG through western blotting assay which suggested a good immunogenicity of pUL55. Dilution method and gradient dialysis were used to restore the natural structure of denatured pUL55. SDS-PAGE and western blotting analysis indicated that the renatured pUL55 obtained higher purity and immunogenicity which was more suitable for producing specific polyclonal antiserum of pUL55.
The obtained rabbit polyclonal UL55 IgG in our work was purified using ammonium sulfate precipitation and High-Q anion-exchange chromatography. SDS-PAGE analysis of the extractive anti-pUL55 IgG detected two expected bands about 55 KDa and 25 KDa respectively. The refolded pUL55 was used to recognize the extractive anti-pUL55 IgG by western blotting assay. These results indicated that the renatured pUL55 has induced a strong immunological response and the prepared antiserum had a high level of specificity. It can be widely used for identification features of DEV UL55 gene product. The titer of agar diffusion reaction reached 1:16 which suggested the extractive anti-pUL55 IgG was specific and sensitive to pUL55. Moreover, the determined titers of Viral neutralization test demonstrated that pUL55 can neutralized DEV and anti-DEV infection, also has the potential to produce subunit vaccines.
Kinetics of UL55 expression in DEV infected DEFs was determined by western blotting. Results suggested the DEV pUL55 became detectable as early as 8 h p.i, increased in amount and reached it highest level at 24 h p.i. No appreciable protein was detected until 60 h p.i. The DEV UL55 protein existed in infedcted cells almost throughout the viral replication cycle. In the temporally regulated cascade of herpesvirus gene expression, the products of herpesvirus genes has been divided into three types according to the transcription conditions of HSV-1, PRV, HCMV. Proteins encoded by immediate-early (IE) and early (E) genes were supposed to be expressed firstly which might be involved in virus replication. The following expressed proteins were structual proteins of virus encoded late(L) genes which were further subdivided into two categories as leaky-late (γ1) or strict-late (γ2). The last kind of proteins were some nonessential proteins encoded by optional genes. To our knowledge, the protein kinase pUS3 and dUT-Pase wich were first detected at 2 h.p.i. and 4 h p.i. respectively has been defined as immediate-early products. By contrast, the pUL31 and pUL51 of DEV were classified to late gene products since they were first detected at 6 h.p.i. and 8 h.p.i, respectively. Consequently, the pUL55 was concluded to be the product of a late gene and might be a component of DEV virions. Researches about HSV-2 UL55 gene product in infected cells suggestted the pUL55 protein was synthesized as a γ2 gene but not a stable component of HSV-2 virions.
Viruses use the host synthetic machinery for replication. Viral proteins need to be targeted to the appropriate intracellular compartments of the host cell to fulfill their roles. Regional distribution of protein in cells will influence the procedures of protein folding, polymn and post-transcriptional modification. Then further affect the fuctions of cell. Only if the synthetic protein be transformed into specific organelle did the vital movements working orderly. Any deviation of location will have significant impacts on functions even the vital movement of cells. Proteins which merely located in nucleus are expected to participate the metabolic processes of DNA or RNA in cells. Otherwise, the proteins distributed in cytoplasm or cytolemma have nothing to do with the above procedures. Study the intracellular location of proteins will increase our understanding of the role of these proteins in host cells and could also be useful for the design of improved therapeutic interventions.
Previously research indicated that the indirect immunofluorescence experiments was a useful method for subcellular location of protein in infected cells. In fact, it is a specific, sensitive and rapid antigen-antibody binding reaction. In our research, we found the location of DEV pUL55 in infected cells was dynamic changes during the life cycle of DEFs. That probably means the pUL55 has an important realationship with the propagation of DEV in DEFs. Results in Figure 10 to Figure 14 suggested the pUL55 was predominantly located in cytoplasm as the computational analysis predicted (60.9% into cytoplasmic), and small amount of it within nuclear. It started to expression in cytoplasm as early as 5.5 h p.i, then diffusion to cytoplasm and gradually distributed near the periphery of the nucleus between 11 h p.i and 35 h p.i. After that, the fluorescence granules clustered to speckled structures and distributed dominantly in the juxtanuclear region from 40 h p.i. At last, the fluorescence diminished since 54 h p.i that suggested the intracelluar location variation of pUL55 might due to the place transformation of protein synthesis and its function exertion. It was presumed that the pUL55 might be synthesized in cytoplasm initially then transformed nearby the periphery of the nucleus to implement its biologic functions. According to previous report, HSV-2 UL55 was located within and near the periphery of nucleus and abutted on and partially overlapped the capsid protein ICP35 which would coalesced VP5, VP19c at late times p.i and located at the periphery of large globular structures composed of proteins involved in DNA replication. Thus, the pUL55 located nearby the perinucler space to pariticipate in the package of virus. When packaged viurs DNA which has been wrappered by ICP35 and its neucleocapsid aggregates transformed nearby, the synthesized pUL55 combined to it as a tegument component or something. However, it might participate in package through some unkown mechanism instead of to be a component. Besides, the distribution of fluorescence transformed once again go along with the variation of DEV viron in infected cells since 35 h p.i can be explained to be related to the propagation of virus in DEFs and cytopathic mechanism. The fuloresence structures gradually diminished to shed off afterwards probably due to the maturity, egress and release of viurs according to the acceptable propagation pattern of DEV in host cells. Apart from that, pUL55 became undetectable probably because it is a low abandance protein in packaged virons or it is not a stable component of DEV virions. Of course, the above assumptions about pUL55 and its mechanism of involving in DEV propagation need to be determined in future studies.
Electron microscopic characterization of duck plague virus suggested the initial progeny virus nuecleocapsids are detectable since 12 h p.i and the mature virus was observed at 24 h p.i. The initial 6 h are latency period of DEV. In our research, pUL55 was firstly detected at 5.5 h p.i which was probably produced by parental viruses since pUL55 has been designated to be a late gene according to previrously report and dynamic expression of pUL55 we had investigated above. The fluorescence granules repesented pUL55 were clusterd to peak at 22.5 h p.i corresponding to the mature time of DEV and the dynamic distribution of pUL55 in cells at 24 h p.i basically. After that, fluorescence became weak gradually due to the release of mature DEV.
In this work, the recombinant plasmid pET32a(+)/UL55 was constructed successfully for expression in prokaryotic system. The purified and renatured recombinant pUL55, which was recognized well with anti-DEV serum, was used for preparation of specific anti-pUL55 serum. Viral neutralization test demonstrated that the pUL55 has the potential to produce subunit vaccines, and possesses the functions of neutralizing DEV and anti-DEV infection. The determined anti-pUL55 serum was used for characterization of pUL55 by Western blotting assay and indrect immunofluorescence. As a result, we found the expression of this gene appeared at the late stage of infection in infected DEFs and pUL55 was predominantly located in cytoplasm and traces of it in nuclear. pUL55 participated the assembly and maturation procedures of virus in some uncertain way. Characterization of pUL55 gave some insights of this gene and DEV investigation. However, further researches about this gene are expected to give more evidence in future.
The research was supported by Changjiang Scholars and Innovative Research Team in University (PCSIRT0848), China Agricultural Research System (CARS-43-8) and China 973 program (2011CB111606).
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