Study On UL13 Protein Kinase Activity And Nuclear Localization Characteristics Of Duck Enteritis Virus

Duck enteritis virus belonged to Mardivirus of Alphaherpesvirinae in Herpesviridae. The protein for DEV UL13 gene coding is one of the family members in Conserved herpesvirus protein kinase (CHPK). According to related reports of the DEV UL13 homologous protein, CHPK show various functions in the total process of the replication of the virus. It can autophosphorylate and phosphorylate the various viruses and host cell proteins, and involve in the viral replication, nucleocapsid and virus Assembly, escape the interferon reaction and other various viruses'physiological activities. At present, there are no any reports on DEV UL13 protein kinase. This paper mainly focuses on the discovery on new information such as location and positioning signal as well as nuclear-import mechanism on character of DEV UL13 gene and its coding protein as well as protein kinase activity in the cells, impact on the nucleation function, relationship of the nuclear localization signal and kinase activity, so as to provide the base material and reference basis for the further study. The main study contents are as follows:1 Bioinformatics analysis of UL13 gene in duck enteritis virus strains CHvDEV UL13 gene ORF is 1575 bp and it codes 524 amino acids. The whole peptide chains do not have the signal peptide and transmembrane domain. There are 9 potential glycosylation sites and 34 phosphorylation sites. There are 2 potential nuclear localization signals which are respectively located at the peptide fragment 4-7 and 90-96 amino acid residues.296-305amino acid sites are displayed as the potential nuclear export signal. The codon analysis indicates that the UL 13 gene tends to use the codon with A/T ending. The codon use mode is closest to the yeasty use mode. The secondary structural analysis of DEV UL 13 protein indicates that there are the multiple antigenic determinants which are evenly distributed in the entire peptide chain. The tertiary structure predicates that it is highly similar to the family members in the serine/threonine protein kinase, which is one of the family members in conserved herpesvirus protein kinase (CHPK). The conserved 179 amino acids will play important roles to the maximize activity for DEV UL13 protein kinase by the sequence alignment.2 DEV CHv UL13 gene cloning, prokaryotic expression and preparation of polyclonal antibody for UL13 proteinThe trucated DEV UL13 gene (486-1158 site of UL13 ORF) was cloned into prokaryotic expression vector pET32c (+), and the recombinant expression vector was transformed into the E. coli BL21 (DE) for the induced unsoluble fusion protein with His-tag. The recombinant protein can be recognized by the rabbit's polyclonal antibody specific anti-DEV CHv. This indicates that the expression of DEV UL 13 protein has good reactogenicity. The optimization of the expression conditions was tobe induced 5 hours at 30? with 0.2mM IPTG, and then the affinity chromatography method was used to purify the UL13 protein. Then purified recombinant UL13 protein was immunized rabbits to obtain the rabbit anti- DEV UL13 protein serum. The prepared serum can specifically recognize the UL 13 protein in the DEV infected cells. This indicates that the immunogenicity of DEV UL13 is good.3 Analysis for the gene type, transcription and expression phase of UL13 gene in duck enteritis virus strains CHvOn the basis of the drug inhibition test using the nucleic acid inhibitor ganciclovir and protein inhibitor cycloheximide, firstly confirmed that DEV UL13 gene is an early gene which could express to be independent of the replication of virus. With the ?-actin gene as an internal control, we analyzed the temporal transcription of the DEV UL13 gene by using the real-time fluorescence relative quantitative PCR (qRT-PCR) and found that the UL13 gene was transcribed at 2 h post-infection and the transcriptional level reached peak at 24 h. But the transcriptional level of ?-actin kept at a high level at the initial stage of the virus infection. It decreased after 4 h and the transcriptional level of ?-actin kept at a low level at 24 h post-infection. Seen from the changes on DEV UL13 gene transcription levels relative to host cell ?-actin gene transcription level, UL13 gene was transcribed at 2 h post-infection, the relevant transcription level gradually increased and reached peak at 48 h post-infection, then it gradually decreased. The western-blot experiment indicated that UL13 protein could be detected at 4 h post-infection, and the expression level reached peak at 24 h post-infection and significantly decreased at 60 h post-infection. The expression level of DEV UL13 gene was corresponding to the dynamic changes of the transcriptional level.4 Study on UL13 protein kinase activity of Duck enteritis virusWe expressed DEV UL13 protein and DEV AUL139(K179M) protein which was the mutant of UL13 for the conserved and important 179 lysine replaced by methionine by using baculovirus insect cell expression system, and compared the differences of the in vitro kinase activity between DEV UL13protein and DEV ?UL139(K179M) protein, and then determined that the optimum enzymatic activity conditions of DEV UL13 in vitro were at 50mM Mg2+ existence, pH7.2,37?. Meanwhile, we purified UL13 potential substrate protein of, glycoprotein gE and Us3 protein kinase by virus coding. We firstly confirmed that the Us3 protein could be phosphorylated by DEV UL13 protein by vitro kinase reaction. In order to further determine the phosphorylation capacity of UL13 protein to Us3 protein, and explore the impact of the UL13 protein kinase activity to virus replication, we successfully constructed the conserved and important 179 lysine replaced by methionine of UL13mutant virus DEV CHv-BACAUL13(K179M) and back mutation virus DEV CHv-BACAUL13(K179M) by using DEV infectious clone platform newly established in our laboratory. By the comparison of the biological characteristics in vitro among DEV CHv-BACAUL13(K179M) mutant virus, back mutation virus DEV CHv-BACAUL13(K179M) R and parental virus DEV CHv-BAC, we found that the virus replication capacity and plaque formation of DEV CHv-BACAUL13(K179M) mutant virus was poorer than the parental virus and back mutation virus. This indicated that UL13 protein kinase activity maight result in the impact on the virus replication. By using immunoblotting method, we determined that the DEV UL13 protein could phosphorlate Us3 protein in the infection-cells.5 Exploration of Duck enteritis virus UL13 protein located in nuclearWe found that UL13 protein of DEV distributed both in nucleus and cytoplasm in infection-cell via indirect immunofluorescence assay (IFA).We constructed series UL13 recombinant plasmid with predicted NLSs deleted to transfect Duck embryo fibroblasts (DEFs), and the result shown that the amount of proteins were significantly decreased than wild type UL13 in nucleus. Then, we constructed the plasmid expressing potential NLSs with GFP and transfected it into DEFs, the result showed that the amount of GFP located in nuclear significantly increased, when compared to the plasmid without NLSs. Also, we proved that this nuclear importing was a classical importin ?/?-dependent process by ivermectin addition. In order to further determine the function that UL13 protein played in nucleus, we successfully constructed the mutant virus with NLS of UL13 deletion, DEV CHv-BAC UL13-ANLS by using similar methods of constructing CHv-BACAUL13(K179M) mutant virus. By the comparison of the biological characteristics in vitro between DEV CHv-BAC UL13-ANLS mutant virus and parental virus DEV CHv-BAC, we found that the virus replication capacity and plaque formation of DEV CHv-BAC UL13-ANLS mutant virus was nearly the same with the parental virus. This indicated that the roles of UL13 protein played in nucleus maight not be related with virus replication, and we also proved that UL13protein with NLS deletion could also phosphorlate Us3 protein in the infection-cells.6 Construction of Duck enteritis virus UL13 deletion strain and its molecular characteristic researchWe successfully constructed the UL13 mostly/partial deletion mutant virus DEV CHv-BAC?UL13, DEV CHv-BAC?UL13-1 and DEV CHv-BAC?UL13-2 by using DEV infectious clone platform. By the comparison of the biological characteristics in vitro among these three mutants and parental virus DEV CHv-BAC, we found that the virus replication capacity and plaque formation of these three mutants was poorer than the parental virus, especially DEV CHv-BAC?UL13-2 mutant. This indicated that UL13 protein played important roles on the virus replication.