Studies On The Function Of Duck Plague Virus UL49Gene

1. Bioinformatic analysis of UL27gene of Duck Plague Virus. In this paper, we conducted a detailed bioinformatic analysis of VP22protein. Our results showing that the DPV VP22protein has a molecular weight of27864.575Da, its pHi is computed to be9.795. The contents of Arg, Ala Ser, Asp and Thr were11.85%,11.85%,11.06%,7.11%and7.11%, respectively. For DPV VP22protein, the carboxyl terminus (126aa～191aa) is highly conserved. Similarity comparison and phylogenetic analysis showed that DPV aligns into the Alphaherpesvirus subfamily and is closely related to MDV. Two N-glycosylation sites, three cAMP-and cGMP-dependent protein kinase phosphorylation sites, nine protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites, two N-myristoylation sites, three Amidation sites were determined by PROSCAN. YLoc predicted that the DPV VP22protein sequence is located in the nucleus, cytoplasm and mitochondria with a probability of87.2%,12.7%and0.1%, respectively. Our results may be useful in future studies to elucidate the biological functions of VP22during DPV infection.2. Cloning, prokaryotic expression, purification and polyclonal antibody preparation of DPV UL49gene. The full-length UL49gene of DPV was amplified with a pair of primer. This product was cloned into the multiple cloning site of the pMD19-T vector. For recombinant protein production, a prokaryotic expression vector pET28a(+) was used. pMD19-UL49was digested with BamH Ⅰ and Hind Ⅲ and the UL49sequense was inserted into the BamH Ⅰ/Hind Ⅲ site of pET-28a(+), which is capable of producing recombinant protein with an N-terminal6xHis tag. The result was designated pET28a-UL49and transformed into E. coli BL21(DE3) for recombinant protein production by calcium chloride method. For optimizing protein expression, cells were harvested4h after induction by0.6mM IPTG at37℃may prove optimal for the yield of soluble protein. For the production of the polyclonal rabbit anti-VP22serum, a adult New Zealand white rabbit was inoculated four times with VP22protein. The rabbit was bled7to10days after the final booster, then it was killed and the IgG fraction of the serum was isolated by ammonium sulfate precipitation. Then, using a DEAE-Sepharose column, the IgG fraction was purified by ion-exchange column chromatography. These results will provide a basis for further analysis of the DPV VP22protein. Western blots shows that the rabbit anti-VP22polyclonal antibody has good specificity.3. Subcellular localization of Duck Plague Virus protein VP22. Indirect immunofluorescence tests revealed that the VP22antigens is detectable as early as6h postinfection, it exists predominantly in the cytoplasm and perinuclear region of DPV infected DEF in a diffuse fluorescent pattern early in infection, then migrates to and accumulates in the nucleus at12hpi, virtually every cell in the culture exhibited bright nucleus fluorescence at36hpi. At late stages of infection, cells expressing fluorescence dramatically reduced due to plaque famation at48hpi and72hpi. VP22found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear. Results of immunofluorescence studies show that the VP22recombinant protein can be detected in DEF after4h incubation in MEM, exhibiting pronounced accumulation in the cytoplasm, but failed migrates to and accumulates in the nucleus, virtually every cell in the culture exhibited bright cytoplasm fluorescence at36h. These results indicate that purified VP22recombinant protein retains the import property.4. Transcription characteristics and expression kinetics analysis of Duck Plague Virus UL49gene. In this study, we determined the transcription characteristics and expression kinetics of DPV UL49gene by FQ-PCR and Western blot method. The results showed that DPV UL49gene transcripts appeared at1hpi, the relative expression level was at a low level in the first12hpi, and VP22protein can be detected at24hpi, and then the transcripts and VP22protein were peaked at72-84hpi, thereafter both of them declined.5. Identification of the nuclear localization signal of Duck Plague Virus VP22protein. In order to mapped the functions of DPV VP22to specific regions in the polypeptide, we constructed a series of deletion constructs of VP22tagged by the GFP. The DPV VP22derivatives was transfected into DEF cells as described in Materials and Methods, and the subcellular localization pattern of the fusion proteins were examined using a Nikon Eclipse TE2000-U Inverted Microscope or a Nikon ECLIPSE80i fluorescence microscope. In this report, we demonstrate for the first time that (1) the nuclear localization of VP22is independent of other viral proteins,(2) VP22does not have a classical NLS, but a89-amino acid sequence from165aa to253aa within the carboxyl terminus was sufficient for nuclear localization.(3) the nuclear localization of VP22is independent of fixing agent.6. The determination of RNA interference target for DPV UL49gene and its transcriptive effect to glycoprotein genes. According to the DPV UL49gene sequence,4shRNA were designed and constructed in pGPU6plasmids by Shanghai GenePharma Co.,Ltd.4RNAi plasmids and VP22a-GFP were cotransfected in DEF cells using LipofectamineTM2000Reagent, the results showed that VP22-505and VP22-555have inhibitive effect. Relative quantitative real-time RT-PCR analysis of UL49mRNA levels throughout DPV infections showed the "5-GCTGTTGGAGATTGCCAATGA-3" sequence (505bp～525bp) can be seen as the best target spot. When DEF cells were transfected with VP22-505expression vectors and then infected with DPV, the transcriptional effect of UL49, gB, gD and gE can be severely down-regulated by VP22-505.