Expression and characterization of the UL31 protein from duck enteritis virus
- Wei Xie1,
- Anchun Cheng†1, 2Email author,
- Mingshu Wang†1, 2,
- Hua Chang2,
- Dekang Zhu1, 2,
- Qihui Luo2,
- Renyong Jia2 and
- Xiaoyue Chen2
© Xie et al; licensee BioMed Central Ltd. 2009
Received: 28 December 2008
Accepted: 10 February 2009
Published: 10 February 2009
Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product.
The entire ORF of the UL31 was cloned into pET 32a (+) prokaryotic expression vector. Escherichia coli BL21(DE3) competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa) was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs.
In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the Alpha herpesvirus subfamily. Furthermore, in vivo experiments with ducks infected with UL31-defective isolates of DEV will also be of importance in order to assess the possible role of the UL31 protein in viral pathogenesis. These properties of the UL31 protein provide a prerequisite for further functional analysis of this gene.
Duck virus enteritis (DVE) is an acute and contagious disease of birds from the order Anseriformes (ducks, geese, and swans) [1–3]. The causative agent of the DVE is Duck enteritis virus (DEV), a member of the subfamily Alphaherpesvirinae . As with many other herpesviruses, DVE can establish inapparent infections in birds that survive exposure to it, a state referred to as latency . This makes the disease difficult to monitor and control. The genome of DEV is composed of a linear, double stranded DNA and the G+C content is 64.3%, higher than any other reported avian herpesvirus in the subfamily Alphaherpesvirinae . There has been little information about the molecular characteristics of DEV since the disease was report in 1926. Although the molecular structure of the genome has not been reported, the DEV genomic library was successfully constructed in our laboratory .
During lytic infection, many herpesvirus proteins are involved in the early steps of viral maturely at the nuclear envelope, which include the UL31 of Herps simplex virus (HSV) and Pseudorabies virus (PRV) [8–11]. The UL31 protein of HSV-1 is a nuclear matrix-associated phosphoprotein stabilized by its interaction with the UL34 protein [12, 13]. The two proteins interact to form a complex colocalized at the nuclear rim of infected cells, and become incorporated into virions during envelopment at the inner nuclear membrane [13–15]. With many similarities and a few differences, accumulating evidence indicates that the UL31 protein and its homology play similar roles in nuclear egress of Alpha-, Beta-, and Grammherpesviruses [8, 14, 16–20]. However, there is no report on the identification and characterization of the UL31 gene product of DEV.
In the present study, the UL31 gene was amplified from the genome of DEV and successfully expressed in a prokaryotic expression system. We prepared polyclonal antiserum which allowed identifying and characterizing the UL31 gene product of DEV. We found that the UL31 gene was transcribed most abundantly during the late phase of replication, and the UL31 protein was approximately 35 kDa and widespread speckled structures in the nuclei of infected cells, but was not detectable in purified virions. In the DEV-infected duck tissues, the UL31 antigen was primarily located in the cells of immunological organs and digestive organs. These properties of the UL31 protein provide a prerequisite for further functional analysis of this gene.
Results and discussion
Predicted features of the UL31 ORF
Expression and purification of recombinant UL31
Preparation and specificity of anti-UL31 protein antiserum
To identify the UL31 product, SDS lysates from DEV noninfected and infected DEF cells were collected and immunoblotted with the anti-UL31 polyclonal antibody. As shown in Fig. 4B, UL31 anti-serum recognized a specific band of approximately 35 kDa in infected cell lines (lane 3). However, no signal was present in uninfected cell lines (lane 4). Nucleotide sequence analysis of coding sequences of UL31 predicts a 35.7 kDa basic protein, and thus the molecular mass of the protein reacted with the UL31 antiserum was consistent with that predicted. These results indicate that the 35 kDa protein is the product of the DEV UL31 gene.
UL31 RNA expression in infected cells
Subcellular location of the UL31 product in DEV-infected cells
The UL31 protein was not detected in extracellular virons
Distribution of DEV UL31 antigen in DEV-infected ducks
In this work, the DEV UL31 gene has been successfully expressed in a prokaryotic expression system, and we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the Alpha herpesvirus subfamily. Furthermore, in vivo experiments with ducks infected with UL31-defective isolates of DEV will also be of importance in order to assess the possible role of the UL31 protein in viral pathogenesis.
Cells and viruses
Duck embryo fibroblasts (DEF) were grown in MEM medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL), 100 units/ml penicillin and 100 μg/ml streptomycin and were used throughout this study. DEV CHv strain was a high-virulence field strain isolated from china, obtained from Key Laboratory of Animal Disease and Human Health of Sichuan Province.
Construction of bacterial expression vector
A full-length UL31 gene was amplified by PCR from the genome of DEV CHv-strain, using synthetic oligonucleotide UL31f (5'-AAAGAATTCATGAGCCAGACCCAACCCCCG) as the forward primer and synthetic oligonucleotide UL31r (5'-TTAGTCGACTACGGCGGAGGAAACTCGTC) as the reverse primer. BamH I and Hind III sites were incorporated into the forward and reverse primers, respectively. The amplicon was cloned into a T/A cloning vector (pMD18-T Simple; TaKaRa). The UL31 sequence was subsequently released by BamH I/Hind III digestion and cloned into the Hind III and BamHI sites of pET 32a(+) (Novagen) in frame with the gene encoding His. The recombinant plasmid (pET-UL31) was confirmed by restriction enzyme digestion and DNA sequencing (TaKaRa)
Expression and purification of UL31-His fusion proteins
The confirmed construct described above was used to chemically transform Escherichia coli BL21(DE3) for expression the UL31 protein. For production of UL31-His fusion protein, 100 μl of fresh stationary-phase culture was inoculated into 10 ml of Luria broth (LB) supplemented with 50 μg/ml ampicillin (Sigma). To optimize expression, the bacterial culture was grown at 37°C until the optical density at 595 nm was 0.5, at which time protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The culture was shaken at 210 rpm at 37°C for 3 h in a 100 ml Erlenmeyer flask. After induction, cells were lysed in 2× sample buffer (0.1 M Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 0.1 M DTT) and analyzed by SDS-PAGE . The recombinant His-tagged proteins were purified by nickel affinity chromatography according to the manufacturer's protocol (Bio-Rad), and analyzed by SDS-PAGE.
Generation of polyclonal antisera in rabbits
For the preparation of polyclonal antibodies, male rabbits were immunized first with 0.5 mg of E. coli-expressed 6× His-tagged UL31 proteins emulsified in Freund's complete adjuvant. Inoculations were subcutaneous injections on the shaven back. Freund's incomplete adjuvant and 1 mg of purified fusion protein were used for subsequent boots. Three booster injections were given each at 1-week intervals after primary injection. Eighteen days after the last boot, blood was collected from an ear vessel. Then, sera were collected and stored at -80°C.
To identify and characterize the DEV UL31 product, DEF, mock infected or infected with DEV, were harvested by centrifugation, washed once with PBS, and resuspended in PBS-1%Triton-2 M urea and briefly sonicated. Then, samples were denatured and resolved on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane by standard procedures . For immunodetection, the membranes were blocked in 5% nonfat dry milk in PBS-T (0.2% Tween 20 in PBS, PH 7.4) for 1 h. The membranes were then washed three times with PBS-T and incubated with diluted rabbit anti-UL31 (1:200) sera for 1 h at 37°C. After three washes with PBS-T, the membranes were incubated with horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG) (Amersham) and specific bands were detected using an enhanced chemiluminescence (ECL system) according to the manufacturer's instructions (Amersham).
Determination of mRNA expression of UL31 in infected cells
The levels of the mRNA transcripts of UL31 were determined by reverse transcriptase polymerase chain reaction (RT-PCR) on total RNA, extracted from uninfected or DEV-infected cells at different times p.i. (6, 12, 24, 36, 48, 60 and 72 h), using the Total RNA Isolation System (TaKaRa). The concentration of RNA was determined by measuring A260, and the purity was checked by the A260/A280 ratio (greater than 1.8). Purified RNA was treated with DNAase I (TaKaRa) and 2 μg RNA was used as template for RT-PCR. The PCR primers for UL31 cDNA and β-actin cDNA are: UL31 f (5'-GTTGCTGCCCAG TATGTT-3') and UL31 r (5'-GTCGGATGCTGCTTGTAT-3'); β-actin f (5'-CCGGGCATCGCTGA CA-3') and β-actin r (5'-GGATTCATCATACTCCTGC TTGCT-3'). cDNA equivalent of 5 ng original RNA was used in PCR. β-actin mRNA expression was determined using the same amount of cDNA as an RNA-competence control.
Indirect immunofluorescence assays of infected cells
The DEV UL31 production location in intracellular was analyzed by Indirect immunofluorescence. DEF cells were seeded on sterile coverslips and were mock or infected with DEV. At 36 h postinfection, cells were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min at 25°C and with 0.2%(v/v) TrionX-100 in PBS for an additional 10 min at 25°C to allow permeabilization. Following several washes in PBS, cells were blocked in 5% bovine serum albumin (BSA) in PBS for 1 h at 37°C. After, The cells were reacted with rabbit anti-UL31 serum diluted 1: 200 in PBS containing 0.1% BSA for overnight at 4°C, washed three times in PBS and then reacted with 1: 100 dilution of FITC-conjugated goat anti-rabbit immunoglobulin in PBS containing 0.1% BSA for 1 h at 37°C. The cell nuclei were visualized by DAPI counter-staining (5 μg/ml, Beyotime). Fluorescent images were viewed and recorded with the Bio-Rad MRC 1024 imaging system.
Biochemical characterization of extracellular virions was performed by precipitating viruses from infectious supernatants with a polyethylene glycol (PEG)-containing solution (0.5% [wt/vol] PEG 6000 in 5 M NaCl) as described previously [17, 31, 32]. Monolayer of DEF cells were infected with DEV and harvested from the extracellular media at 72 h postinfection by centrifugation at 10,000 × g for 20 min. To purify intracellular virions, lytically induced cells were extensively washed and sequentially frozen in a dry ice bath and thawed at 37°C three times. Cells were spun down at 5,000 × g for 10 min, and supernatants were filtered with a 0.45-μm-pore-size filter. Viruses present in these supernatants were further PEG precipitated as described for extracellular virions. Purified virions were analyzed by Western blotting.
Immunofluorescence image analysis of UL31 antigen distribution
To monitor the UL31 antigen distribution in DEV infected ducks, thirty-day-old ducks (DEV free) were used. The ducks were divided into 2 groups (A and B): Group B (11 ducks) was mock-infected with PBS by intramuscular injection; Group A (22 ducks) was infected with DEV (half of LD50) by intramuscular injection. After 4 d post-infection, different tissues were obtained and immediately treated with 4% formaldehyde for 24 h, and then embedded in paraffin.
Four-μm thick histological sections were cut from each tissue, mounted, and baked. They were then deparaffinized and rehydrated in PBS. For antigen retrieval, the sections were treated with 0.01 mol/L citrate buffer solution (pH 6.0) for 10 min in a microwave oven. Nonspecific binding was prevented by treating the sections with 5% bovine serum albumin (BSA) at 37°C for 30 min. The sections were then treated with 1:100 diluted anti-UL31 sera for 1 h at 37°C and washed with PBS. Then, they were treated with FITC – conjugated goat anti-rabbit IgG (1:100). Slides were washed in three changes of PBS, counterstained lightly with Evans blue (EB) (0.01% for 3 min), dehydrated, and coverslipped. Images were examined under the Bio-Rad MRC 1024 imaging system.
The research were supported by the National Natural Science Foundation of China (30771598), Changjiang Scholars and Innovative Research Team in University(PCSIRT0853), New Century Excellent Talents program in University (NCET-06-0818), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (706050), the Cultivation Fund of the Key Scientific and Technical Innovation Project, department of Education of Sichuan Province (07ZZ028), Sichuan Province Outstanding Youths Fund (05ZQ026-038/07ZQ026-132), Sichuan Province Basic esearch Program (05JY029-109/05JY029-003/07JY029-016/07JY029-017/2008JO0003/2008JY0100/2008JY010 2) and Program for Key Disciplines Construction of Sichuan Province (SZD0418).
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