| Newcastle disease virus (NDV) is an economically important avian virus that may result in substantial loss to the poultry industry. Newcastle disease virus (NDV) belongs to the new genus Avulavirus within the family Paramyxoviridae. The non-segmented negative-sense single-stranded RNA genome contains six major genes that encode the structural proteins in the order 3`-NP-P-M-F-HN-L-5`as well as two non-structural proteins, W and V. The P gene encodes three proteins via mRNA editing, and P protein is encoded by an unedited transcript one. The P protein is known as the viral RNA polymerase subunits involved in both transcription and replication during the virus life cycle, and it is highly phosphorylated in comparison to other viral proteins. In that case, it was called Phosphoprotein. Phosphorylation of P proteins in several negative strand RNA viruses by specific cellular kinases was found to be required for P protein function.But the functional significance of the P protein phosphorylation is not clear and this in part may stem from the lack of information on the precise location of the phosphorylation site(s) in the P protein.In current study, to identify the functional phosphorylation sites in Phosphoprotein of NDV, and investigate the role of phosphorylation by cellular kinase in the regulation of P protein activities and in the formation of active transcription and replication complexes.The potential phosphorylation sites within the P protein is predicted using the bioinformatics software and online databases.To confirm the role of cellular kinase which targeted for the sites, the effect of specific inhibitors on NDV propagation were detected. The function of phosphorylation status of P protein was analysed using the La Sota minigenome replication/transcription system. A series of P mutants at different sites were obtained by overlap PCR. Replacing P helper-plasmid with various mutant P plasmids, the genome, antigenome and mRNAs level was quantitated by real-time RT-PCR.1. Mapping the potential and functional phosphorylation sites in Phosphoprotein of NDVIn the study, we selected 28 entire genome sequences of NDV that had published in GenBank, and the P gene were analyzed by amino acid sequences alignment. Since the phosphorylation of the P proteins of nonsegmented negative strand RNA virus remains stringently conserved, we found 22 Serine sites, 7 threonine sites and a tyrosine sites were particularly conserved in the strains. Seven potential phosphorylation sites among these conserved residues were mapped and located in consensus sequences characteristic for protein kinase PKC by using the online database NetPhosK 1.0 Server and Netphos 2.0 Server. It seems to be a degree of consistency in terms of the cellular kinases involved in protein phosphorylation as reported for other Paramyxoviruses.2. The role of phosphokinase from host cells in the course of infection of NDVIn order to explore the celluar phosphorylation pathways involved in the course of infection of Newcastle Disease Virus (NDV), the effect of diverse activators and inhibitors of phosphokinase on NDV propagation were detected in this study. After treatment with activators or inhibitors to phosphokinase, DF1 were infected with Herts/33,a velogenic NDV strain. The titre of virus in the supernatant was successively detected every 8 hours. The propagation of NDV was inhibited when pretreated with the inhibitor of proteinkinase C, staurosporine, while this effect of propagation delay can be blocked by the activator of proteinkinase C. To confirm the role of proteinkinase C phosphorylation pathways in the course of NDV,a propagation rate of the virus in the cells pretreated with staurosporine was figured out. The results suggested that staurosporine inhibit the virus infection during the drug concentration from 20nmol/L to 200nmol/L.The more concentrated drugs were used, and the more obvious propagation delay were found. To conclude, this study shows that the proteinkinase C will be paly a key role in the course of infection of NDV in host cells.3. The role of phosphorylation status of P protein in replication/transcription of the genomeThe function of phosphorylation status of P protein was analysed using the La Sota minigenome replication/transcription system. A series of P mutants at different sites were obtained by overlap PCR. Replacing P helper-plasmid with various mutant P plasmids resulted in a reduced level of eGFP expression.The genome, antigenome and mRNAs level was quantitated by real-time RT-PCR to show the direct role of P protein phosphorylation on virus RNA synthesis. Since mRNA in the presence of P111 /P111-125/P null is approximately 30% of expression in the presence of WT P, mutant P111 can be considered completely defective in minigenome transcription, whereas the mutant P125 did not show any effect. And the level of genome kept at a stable level in the presence of P mutants, indicates that the phosphorylation of P is dispensable for replication. This novel property supports the view that P protein is a candidate to function as a transcription-replication"switch".The present work provides the first report on the role of phosphorylation of NDV P protein in replication/transcription of the virus genome.And the localization of functional phosphorylation sites in Phosphoprotein of Newcastle Disease Virus is a attractive targets for the development of anti-viral drugs. |
