Study On The Characteristics And Basic Functions Of Duck Plague Virus CHv Strain UL54 Gene And Its Encoded Protein

Duck plague virus(DPV)belongs to the herpes virus family,a-herpes virus subfamily,Marek's virus genus.It leads to ducks,geese and other waterfowl infection,and results in serious morbidity and mortality.Compared with other herpes viruses,molecular biology research of DPV started late but rapidly.Though majority of gene research are ongoing,the specific role of UL54 gene and its encoded protein in the DPV pathogenesis is still unknown.Therefore,the main purpose of this paper is to help explain the pathogenesis of DPV by analyzing the characteristics and basic functions of DPV UL54 gene and its encoded protein.In this paper,bioinformatics analysis and polyclonal antibody preparation of DPV UL54 were carried out;transcriptional phase and expression phase were analyzed by Real-time fluorescence quantitative PCR and Western blotting respectively;followed the intracellular localization of UL54 protein was analyzed by indirect immunofluorescence assay(IFA);then,the cytoplasmic shuttle activity and its functional region of UL54 protein were analyzed by performing interspecific heterokaryon assay;UL54 gene deletion and revertant strains based on BAC were constructed and the basic biological characteristics,including growth curve,plaque formation and virus Genomic DNA copy number,were evaluated;then effects of DPV UL54 on the mRNA expression of UL19,UL30,UL48,gC,gD,gK at different time points were studied by Real-time fluorescence quantitative PCR,and the roles of UL54 in transcription,mRNA export and translation were studied based on nuclear runoff assay,direct fluorescence in situ hybridization test and the ribosomal-nascent peptide complex(RNC)respectively;finally,the effect of DPV UL54 on DEF cell apoptosis was studied in the ways of DAPI staining,HE staining,Annexin V-FITC/PI double staining,analysis of relative expression level and activity of Caspase-3.The results indicated that:? the identified ORF of DPV UL54 which composed of 1377 bp nucleotides encoded 458 amino acids with 18 epitopes,and with no signal peptide and transmembrane region;codon usage pattern of DPV UL54 was close to the E.coli,and all of the amino acids,excluding Met,Trp and Glu,have a high level of diversity in codon usage and bias towards the codons with A/T at the third codon position;the UL54 protein is mainly located in the nucleus,and contains a nuclear export signal and multiple nuclear localization signals;? DPV UL54 belongs to the immediate early gene,and the molecular weight of the protein is about 57.0 KDa;After infection,the transcripts and protein could be detected at 0.5 h and 2 h respectively,and the peak are reached at 24 h;the dynamic changes of transcription and expression are basically the same;? the UL54 protein was mainly located in the cytoplasm in early times,gradually transferred to the nucleus as the infection progresses,fully entered into the nucleus at 48 h,and became weaker following cytoplasmic disintegration subsequently;? DPV UL54 protein shows a nuclear shuttle activity and contains functional NES,which is sensitive to lysopromycin B(LMB)and has no significant effect on the localization of UL54 protein;It also contains multiple functional NLS,which affects the UL54 protein itself localization;Furthermore,the nuclear location of UL54 protein plays an important role in virus replication,plaque formation,genomic DNA replication and viral gene mRNA expression;? DPV UL54 is not required for viral replication but can promote viral proliferation,plaque formation and viral genomic DNA replication;? DPV UL54 positively and negatively regulates the mRNA expression of viral genes including UL19,UL30,UL48,gC,gD and gK;it mainly inhibits the UL19 mRNA expression,promotes the UL30 and gC during infection,inhibits UL48 and gD gene is early times but promotes in later times,and promoted or inhibited gK in the early stage of infection;? DPV UL54 promote the transcription of all the target genes but the early UL30;? DPV UL54 help the UL30 mRNA export from nucleus;? In the early stages of infection,DPV UL54 inhibits the translation of the target genes,but promotes in later stages;? DPV UL54 had no effect on the DEF cell apoptosis and DPV-induced apoptosis of DEF cells.In this paper,the DPV UL54 gene and its encoded protein were systematically studied.In particular,the shuttling property of UL54 protein and the regulation of viral gene expression were demonstrated.We clarified the roles of UL54 in DPV pathogenesis from the molecular level with great theoretical and practical value.