Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M.
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SUBMITTER: Lin M
PROVIDER: S-EPMC4178140 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M.
Lin Meng M Jia Renyong R Wang Mingshu M Gao Xinghong X Zhu Dekang D Chen Shun S Liu Mafeng M Yin Zhongqiong Z Wang Yin Y Chen Xiaoyue X Cheng Anchun A
Journal of veterinary science 20140402 3
The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/ glycoprotein M (gM) ...[more]
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Project description:BackgroundDuck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited.MethodsThe DEV UL41 gene was cloned into the pET32a(+) vector and expressed in a prokaryotic expression system. Antiserum was raised against a bacterially expressed UL41-His fusion protein for further experiments. Transcription was quantified and UL41 protein expression levels were determined in DEV-infected cells at different time points by RT-qPCR and western blotting, respectively. DEV virions were purified by sucrose gradient centrifugation and analyzed by mass spectrometry to identify protein content. We confirmed the DEV UL41 gene kinetic class using a pharmacological test. IFA was used to analyze the intracellular localization of pUL41.ResultsThe recombinant expression plasmid, pET-32a(+)-UL41, which highly expresses a 76.0 kDa fusion protein, was constructed and expressed in E. coli BL21 (DE3) after induction with 0.2 mM IPTG at 30 °C for 10 h, generating a specific mouse anti-UL41 protein polyclonal antibody. RT-qPCR and western blot analyses revealed that the UL41 transcript number peaked at 36 hpi, and peak protein expression occurred at 48 hpi. The pharmacological test showed that UL41 was a γ2 gene. Mass spectrometry analysis showed that pUL41 was a virion component. IFA results revealed that pUL41 was localized throughout DEV-infected cells but only localized to the cytoplasm of transfected cells. DEV pUL47 translocated pUL41 to the nuclei of DEF cells; this translocation was dependent on predicted pUL47 NLS signals (40-50 aa and 768-777 aa).ConclusionsDEV UL41 is a γ2 gene that encodes a virion structural protein, pUL41 localizes throughout DEV-infected cells but only localizes to the cytoplasm of transfected cells. pUL41 cannot autonomously localize to the nucleus, as this nuclear localization is dependent on predicted DEV pUL47 NLS signals (40-50 aa and 768-777 aa).
| S-EPMC5769551 | biostudies-literature
Project description:BACKGROUND:There is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization. METHODS:Duck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance. RESULTS:The recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a ?2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein. CONCLUSIONS:The DEV US10 gene is a ?2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.
| S-EPMC5607491 | biostudies-literature
Project description:BACKGROUND: Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data. RESULTS: In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to study the transcription characteristic of the DEV UL53 gene. Except detecting the mRNA of DEV UL53 gene, the product gK encoded by UL53 gene was detected through the expression kinetics of UL53 gene by the purified rabbit anti-UL53 protein polyclonal antibodies. Western-blotting and indirect immunofluorescence assays were used to detect gK. From the results of these experiments, the UL53 gene and gK were respectively identified as a late gene and a really late protein. On the other hand, the indirect immunofluorescence assay provided another information that the intracellular localization of DEV gK was mainly distributed in cytoplasm. CONCLUSIONS: By way of conclusions, we conceded that DEV UL53 gene is a really late gene, which is coincident with properties of UL53 homologs from other herpesvirus, such as ILTV(Infectious Laryngotracheitis virus) and HSV-1(Herpes simplex virus type 1). The properties of intracellular localization about gK protein provided a foundation for further functional analysis and further studies will be focused on constructing of the UL53 gene DEV mutant.
| S-EPMC3120784 | biostudies-literature
Project description:BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
| S-EPMC3117846 | biostudies-literature
Project description:BACKGROUND: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells. RESULTS: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. CONCLUSION: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.
| S-EPMC2714536 | biostudies-literature
Project description:BACKGROUND: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. RESULTS: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-?-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. CONCLUSIONS: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.
| S-EPMC3118967 | biostudies-literature
Project description:BackgroundDuck Enteritis Virus (DEV), belonging to the α-herpesvirus subfamily, is a linear double-stranded DNA virus. Glycoprotein H and L (gH and gL), encoded by UL22 and UL1, are conserved in the family of herpesviruses. They play important roles as gH/gL dimers during viral entry into host cells through cell-cell fusion. The interaction between gH and gL has been confirmed in several human herpesviruses, such as Herpes Simplex Virus (HSV), Epstein-Barr virus (EBV) and Human Cytomegalovirus (HCMV). In this paper, we studied the interaction between DEV gH and gL.ResultsRecombinant plasmids pEGFP-N-gH and pDsRED-N-gL were constructed successfully. Expressions of both DEV gH and gL were observed after incubation of COS-7 cells transfected with pEGFP-N-gH and pDsRED-N-gL plasmids after 12 h, respectively. Also, the co-localization of a proportion of the gH and gL was detected in the cytoplasm of COS-7 cells after co-transfection for 24 h. Then, pCMV-Flag-gL and pCMV-Myc-gH recombinant plasmids were constructed and co-transfected into COS-7 cells. It was showed that both gH and gL were tested with positive results through co-immunoprecipitation and Western-blotting.ConclusionsOur results demonstrated not only the co-localization of DEV gH and gL in COS-7 cells, but also the interaction between them. It will provide an insight for the further studies in terms of protein-protein interaction in DEV.
| S-EPMC6114530 | biostudies-literature
Project description:BACKGROUND: Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all Alphaherpesvirus subfamily. However, there is no report on the VP19c protein of duck enteritis virus (DEV). In this study, we expressed the DEV VP19c protein and presented its antigenic properties. Moreover, we developed polyclonal antibody against the VP19c protein and characterized it. METHODS: A recombinant VP19c (rVP19c) and N-terminal were expressed in Escherichia coli (E.coli) and purified by Ni²+-affinity chromatography. The antigenic properties of the recombinant protein were determined by Western blot and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the polyclonal antibodies against the purified recombinant proteins were produced and the titer of polyclonal antibody was determined by ELISA analysis. Finally, the antibody was used to recognize the VP19c in the cells infected with DEV in the immunofluorescence assay. RESULTS: The N-terminally His-tagged rVP19c and rVP19c(N) were produced as inclusion bodies in E. coli strain BL21 (DE3) with molecular weight of about 66 and 46 kDa. Then the proteins were purified to reach the level of homogeneity. Western blot and ELISA analysis that the rVP19c seems to be structurally and antigenically very similar to native VP19c and the N-terminus of VP19c may contain most antigenic linear-epitopes. Furthermore, ELISA analysis demonstrated that the titer of polyclonal antibody was approximately 1:12800, and in the immunofluorescence assay, the antibody was able to recognize the VP19c in the cells infected with DEV. CONCLUSIONS: To our knowledge, this was the first report on basic properties of DEV VP19c protein. In the present study, we obtained a high-level expression of the recombinant VP19c protein as well as high titers of rabbit polyclonal antibody against to VP19c protein. The anti-rVP19c serum was able to detect the VP19c protein in DEV infected cells and the VP19c protein targeted to the nucleus as distinct punctate speckles. This specific polyclonal antibody provides a good tool for further studying structural and functional characterization of DEV VP19c.
| S-EPMC3050827 | biostudies-literature
Project description:BACKGROUND: Previous studies have indicated that the UL16 protein and its homologs from herpesvirus were conserved and played similar roles in viral DNA packaging, virion assembly, budding, and egress. However, there was no report on the UL16 gene product of duck enteritis virus (DEV). In this study, we analyzed the amino acid sequence of UL16 using bioinformatics tools and expressed in Escherichia coli Rosetta (DE3) induced by isopropy1-?-D-thiogalactopyranoside (IPTG). The recombinant protein was produced, purified using a Ni-NTA column and used to generate the polyclonal antibody against UL16. The intracellular distribution of the DEV UL16 product was carried out using indirect immunofluorescence assay. RESULTS: In our study, UL16 gene of DEV was composed of 1089 nucleotides, which encoded 362 amino acids. Multiple sequence alignment suggested that the UL16 gene was highly conserved in herpesvirus family. The UL16 gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli Rossetta (DE3) induced by IPTG. A 60kDa fusion protein band corresponding to the predicted size was produced on the SDS-PAGE, purified using a Ni-NTA column. Anti-UL16 polyclonal sera was prepared by immunizing rabbits, and reacted with a band in the IPTG induced cell lysates with an apparent molecular mass of 60 kDa. In vivo expression of the UL16 protein in DEV infected duck embryo fibroblast cells (DEFs) was localized mostly around perinuclear cytoplasmic area and in cytosol using indirect immunofluorescence assay. CONCLUSIONS: The UL16 gene of DEV was successfully cloned, expressed and detected in DEV infected DEFs for the first time. The UL16 protein localized mostly around perinuclear cytoplasmic area and in cytosol in DEV infected DEFs. DEV UL16 shared high similarity with UL16 family members, indicating that DEV UL16 many has similar function with its homologs. All these results may provide some insight for further research about full characterizations and functions of the DEV UL16.
| S-EPMC3179959 | biostudies-literature
Project description:Purpose:To understand the transcriptome regulator of duck spleen infected with duck enteritis virus (DEV).Methods:50-day-old ducks were inoculated with 100 titer (The TCID50 of DEV was 10-9/0.1mL) and 10-2 titer two different viral titer of DEV in leg muscle for different durations (66 h, 90 h and 114 h) and seronegative control (0 h) were analyzed using next-generation RNA sequencing.Furthermore, the data were validated using quantitative real-time PCR.Results:There were 534, 685 and 580 genes differentially expressed in 100 titer, moreover, 511, 485 and 531 differentially expressed genes (DEGs) were obtained from 10-2 titer for 66 h, 90 h and 114 h, respectively. These genes were mainly involved in functional categories including immune response, extracellular space, heparin binding, oxygen transport, extracellular region, cellular response to interleukin-4, MHC class II protein complex, antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, and pathways such as ribosome, ECM-receptor interaction, cell adhesion molecules, JAk-STAT signaling pathway, PPAR signaling pathway, neuroactive ligand-receptor interaction, phagosome.Conclusions: Different titers of DEV infection can stimulate different biological processes and signaling pathways in the spleen, and regulated the complex biological processes, metabolic and signaling pathways in the process of DEV infection.This transcriptome analysis of duck spleen infected with DEV in different time points is reported for the first time, it laid the foundation for further understanding of interactions between DEV and duck spleen tissue, molecular mechanisms of duck defend against DEV infection, and screening key functional genes.
2020-06-27 | GSE100524 | GEO