J Vet Sci.  2014 Sep;15(3):389-398. 10.4142/jvs.2014.15.3.389.

Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M

• Lin M
• Jia R
• Wang M
• Gao X
• Zhu D
• Chen S
• Liu M
• Yin Z
• Wang Y
• Chen X
• Cheng A

• KMID: 2155622
• DOI: http://doi.org/10.4142/jvs.2014.15.3.389

Abstract

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) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.

Keyword

colocalization; duck enteritis virus; intracellular localization; molecular characterization; UL49.5 protein

MeSH Terms

Animals
Ducks/virology
Genes, Viral/genetics
Mardivirus/*genetics
Membrane Glycoproteins/*genetics
Microscopy, Fluorescence
Phylogeny
Polymerase Chain Reaction/veterinary
Viral Envelope Proteins/*genetics
Membrane Glycoproteins
Viral Envelope Proteins

Figure

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  Fig. 1 Construction of the recombinant plasmids. (A) Construction of the pEGFP-C1/pUL49.5 plasmid. (B) Construction of the pDsRed1-N1/gM plasmid.

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  Fig. 2 Identification of the recombinant plasmids using PCR and restriction enzyme digestion. (A) Identification of the pEGFP-C1/pUL49.5 plasmid. M, DNA marker; Lane 1, products produced by BamHI and HindIII; Lane 2, product produced by BamHI; Lane 3, product produced by HindIII; Lane 4, PCR product. (B) Identification of the pDsRed1-N1/gM plasmid. M, DNA marker; Lane 1, products produced by XhoI and SacII; Lane 2, undigested pDsRed1-N1/gM plasmid; Lane 3, product produced by SacII; Lane 4, PCR product.

• 

  Fig. 3 Phylogenetic analysis and prediction of functional sites in duck enteritis virus (DEV) pUL49.5. (A) The phylogenetic tree generated based on DEV pUL49.5 and gN sequences of 15 reported herpesviruses. (B) Sequence alignment showing evolutionary relationships. Red indicates similarities while blue indicates differences. (C) Predicted signal peptide in DEV pUL49.5 according to SignalP-HMM. (D) Predicted signal peptide in DEV pUL49.5 according to SignalP-NN. "C score" indicates the cleavage site probability, "S score" indicates the possible region of the signal peptide, and the "Y score" is a derivative of the C score representing the cleavage site probability. (E) Predicted phosphorylation sites in DEV pUL49.5.

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  Fig. 4 Prediction of the DEV pUL49.5 structure. (A) Prediction of the primary DEV pUL49.5 structure. The black wire frame represents the transmembrane domain, the hydrophobicity plot is displayed in purple, and the surface probability plot is shown in yellow. (B) Transmembrane domain profile of gN. The extracellular domain is pink, the transmembrane domains are red, and the cytoplasmic domains are blue. (C) Prediction of the DEV pUL49.5 secondary structure. "H" represents the alpha helix, "E" represents the extended strand, and "C" represents the random coil.

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  Fig. 5 Intracellular localization and distribution of DEV pUL49.5 in DEFs. In the first column, green indicates expression of the pUL49.5+EGFP fusion protein. In the second column, blue represents the cell nuclei counter-stained with DAPI. Merged images are shown in the third column. In the fourth column, green represents EGFP expression as a negative control.

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  Fig. 6 Intracellular localization and distribution of DEV gM in DEFs. In the first column, red represents expression of the gM+RFP fusion protein. In the second column, blue represents nuclei counter-stained with DAPI. The third column contains merged images. In the fourth column, red represents the expression of RFP as a negative control.

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  Fig. 7 Colocalization of DEV pUL49.5 and gM in DEFs. In the first column, green represents expression of the pUL49.5+EGFP fusion protein. In the second column, red represents expression of the gM+RFP fusion protein. The third column contains merged images. In the fourth column, green represents the expression of EGFP as a negative control. In the fifth lane, red represents the expression of RFP as a negative control.

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