Identification and Characterization of Duck Plague Virus Glycoprotein C
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Lian et al. Virology Journal 2010, 7:349 http://www.virologyj.com/content/7/1/349 RESEARCH Open Access Identification and characterization of duck plague virus glycoprotein C gene and gene product Bei Lian1, Chao Xu1†, Anchun Cheng1,2,3*†, Mingshu Wang1,2*†, Dekang Zhu1,2, Qihui Luo2, Renyong Jia2, Fengjun Bi 2, Zhengli Chen2, Yi Zhou2, Zexia Yang2, Xiaoyue Chen1,2,3 Abstract Background: Viral envelope proteins have been proposed to play significant roles in the process of viral infection. Results: In this study, an envelope protein gene, gC (NCBI GenBank accession no. EU076811), was expressed and characterized from duck plague virus (DPV), a member of the family herpesviridae. The gene encodes a protein of 432 amino acids with a predicted molecular mass of 45 kDa. Sequence comparisons, multiple alignments and phylogenetic analysis showed that DPV gC has several features common to other identified herpesvirus gC, and was genetically close to the gallid herpervirus. Antibodies raised in rabbits against the pET32a-gC recombinant protein expressed in Escherichia coli BL21 (DE3) recognized a 45-KDa DPV-specific protein from infected duck embryo fibroblast (DEF) cells. Transcriptional and expression analysis, using real-time fluorescent quantitative PCR (FQ-PCR) and Western blot detection, revealed that the transcripts encoding DPV gC and the protein itself appeared late during infection of DEF cells. Immunofluores- cence localization further demonstrated that the gC protein exhibited substantial cytoplasm fluorescence in DPV- infected DEF cells. Conclusions: In this work, the DPV gC protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gC product for the first time. These properties of the gC protein provided a prerequisite for further functional analysis of this gene. Background UL31[7,8], UL35[9,10], UL51[11,12], dUTPase[13], and Duck plague virus (DPV), or duck enteritis virus (DEV), gE[14] gene. However, the key genes and their functions is an important pathogen of ducks, which has caused remain to be elucidated, especially the viral envelope serious losses in commercial duck production in domes- protein genes. Viral envelope proteins are particularly tic and wild waterfowl as a result of mortality, condem- important because of their role in the virus-host rela- nations, and decreased egg production[1]. DPV is tionship, including recognition, attachment and penetra- classified as the subfamily alphaherpesvirinae of the tion of the virus into susceptible cells. In 2006, the DPV family herpesviridae based on the report of the Eighth genomic library was successfully constructed in our International Committee on Taxonomy of Viruses laboratory, and one envelope protein gene, gC (NCBI (ICTV), but has not been grouped into any genus[2]. GenBank accession no. EU076811) was characterized The genome of DPV is composed of a linear, double- [15-18], but the basic properties and biological functions stranded DNA, with 64.3% G+C content which is higher of this envelope protein are not known. than any other reported avian herpesvirus in the sub- gC is a major component of the virion envelope and is family alphaherpesvirinae[3]. To date, more and more proved to be a multifunctional protein. gC homologues DPV genes have been identified, such as UL24[4-6], of herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), and bovine herpesvirus type 1 (BHV-1), is the primary attachment protein, interacting with cell * Correspondence: [email protected]; [email protected] † Contributed equally surface heparan sulfate proteoglycans (HSPG), thus 1Avian Diseases Research Center, College of Veterinary Medicine of Sichuan mediating efficient virus attachment to the cells[19-24]. ’ Agricultural University, Ya an, Sichuan, 625014, China gC of HSV-1, varicella-zoster virus (VZV) and PRV Full list of author information is available at the end of the article © 2010 Lian et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lian et al. Virology Journal 2010, 7:349 Page 2 of 11 http://www.virologyj.com/content/7/1/349 [25-28] is also a major determinant for virulence. In Transcriptional analysis of DPV gC gene case of Marek’s disease virus (MDV), gC is required for FQ-PCRs were used to detect the expression of the DPV gC horizontal transmission, together with US2, UL13 in gene during viral infection. Total RNA was isolated from combination[29]. Furthermore, gC has been demon- mock-or DPV-infected cells at indicated times, and then strated to be a critical immune evasion molecule, and cDNA was synthesized using reverse transcriptase. Aliquots the two glycoproteins, gC and gE, have a synergistic of cDNA at each time point were used as template for real- effect on mediating immune evasion[30,31]. And gC of time PCR reactions containing primers either for gC gene HSV-1and-2,BHV-1,PRV,andEquineherpesvirus or for b-actin. Table 1 presents data from experiments types 1 and 4 (EHV-1 and -4) has been reported to bind where the target (gC) and reference (b-actin) were amplified ΔΔ complement component C3[32-34], thus modulating in separate wells. The 2- Ct method was used to calculate complement activation. Although nonessential for virus relative changes in the gene expression determined from infectivity of cultured cells, gC is a highly antigenic gly- quantitative real-time PCR experiments. As shown in Figure coprotein, of which the importance in eliciting immune 5 the level of gC mRNA had been increasing since 4 hpi, responses has been well documented for many herpes- peaked between 28 and 36 hpi, and then declined. viruses[35-39]. However, whether the product of DPV gC gene shares these functions remains to be Time course expression of DPV gC protein determined. In this experiment, DEF cells were mock infected or To begin addressing questions regarding gC properties infected with DPV, and at 4, 16, 32, 48, 60, 72 hpi, cell or functions, we cloned and expressed the gC gene from suspensions were harvested and lysed in RIPA buffer. DPV in the prokaryotic expression system, raised anti- Equal amounts of cell lysates were resolved by SDS- serumthatrecognizesthegCproteinandrevealedits PAGE, and proteins on the gel were electrophoretically temporal transcription course and subcellular localiza- transferred to PVDF membrane and subjected to Wes- tion in DPV-infected DEF cells. This work might pro- tern blot analysis with rabbit anti-DPV gC serum. The vide a foundation for further studies on the function of result, shown in Figure 6 revealed that a 45 kDa protein DPV gC. was readily detected as early as 4 hpi and seemed to be present at increased levels at 48 hpi. Results Cloning, prokaryotic expression and antigenicity analysis Subcellular localization of the recombinant protein The subcellular localization of gC protein was examined DPVgCgenefromthegenomicDNAwasamplified by indirect immunofluorescence staining. At various and cloned into a T/A cloning vector pMD18-T, gen- times after infection, DEF cells infected with DPV were erating a recombinant cloning plasmid pMD18-T/gC fixed with 4% paraformaldehyde, treated with 5% bovine (Figure 1). The recombinant plasmid was confirmed by serum albumin (BSA) to block nonspecific binding and DNA sequencing, PCR and restriction digestion (Figure reacted with the DPV gC antiserum. Specific fluores- 2a). The gC gene fragment, which was obtained by cence became detectable only in the cytoplasm of digestion of pMD18-T/gC with EcoRI and XhoI, was infected cells as early as 4 hpi. At later times of infec- ligated into the fusion expression vector pET32a(+) tion, the protein converged at the perinuclear region of (Figure 3) and identified by PCR and restriction diges- the cytoplasm. And after 60 hr, fluorescence was gently tion (Figure 2b). After confirmation, a positive clone dispersed (Figure 7). was submitted to DNA sequencing and the result con- firmed that the gC gene was in frame with the N-term- Discussion inal His6 tag within the pET32a(+) multiple cloning In this study we expand our initial observations, in sites (data not shown). Then this recombinant plasmid, which we identified and characterized the DPV gC gene pET32a-gC, was transformed into Escherichia coli and found that it has homologues in some herpesviruses BL21 (DE3) which, following induction with IPTG, sequenced to date. Analysis of the predicted 432 amino expressed large quantities of the pET32a-gC recombi- acid DPV gC protein indicates that it has several fea- nant protein (Figure 4a), and this recombinant protein tures common to other herpesvirus glycoproteins. There was purified by gel and electric elution (Figure 4b). In is a hydrophobic sequence of about 22 amino acids at order to examine the reactivity and specificity of the the amino terminus that corresponds to a signal peptide recombinant fusion protein, Western blot analysis was and a 23-amino-acid hydrophobic sequence near the carried out. As shown in Figure 4b, the anti-DPV carboxy terminus that is predicted to span the mem- serum specifically recognized a 65 kDa band, which brane of the virus envelope. Amino acid sequence com- corresponded to the theoretical molecular mass of parison revealed that DPV gC gene displayed similarities pET32a-gC. of 29.9%, 29.5%, 31.7% to this gene from MeHV-1, Lian et al. Virology Journal 2010, 7:349 Page 3 of 11 http://www.virologyj.com/content/7/1/349 Figure 1 Schematic diagram of gC gene cloned into the pMD18-T cloning vector. MDV1, MDV2, respectively. Further detailed analysis As a first step toward the study of the gC protein, rab- showed that it possessed a Marek’s disease glycoprotein bit polyclonal antiserum specific to this protein were A conserved structural domain between the residues raised using an Escherichia coli BL21-produced recom- 170 and 425, indicating that the DPV gC and its coun- binant gC fusion protein as antigen.