Preliminary Study On The Regulation Of Host YB-1 Protein Mediated By Newcastle Disease Virus Infection

Newcastle disease virus(NDV)is a very important bird virus heavily related with poultry industry,and virulent NDV can cause highly contagious and fatal Newcastle disease(ND)to birds.However,it has been reported recently that NDV replicated in tumor cells,so NDV has been intensively studied because of oncolytic character.The Y-box-binding protein(YB-1)represents the most evolutionarily conserved nucleic-acid-binding protein currently known.YB-1 has a variety of biological functions,including transcription regulation,translation regulation,drug resistance and stress response to extracellular signal.News reports say that the formation of YB-1 was related with the formation of SG(stress granule).In the process of stress response,YB-1 continued to aggregate in SG.Under diverse forms of cell stress such as oxidative stress,hypoxia,nutrient deprivation,or virus infection,the initiation stage of translation is rapidly blocked,and cells can form P-bodies by the nucleating proteins,then further accumulation form stress granules,likely as a mechanism to limit the synthesis of energy-demanding protein.This study was aimed at demonstrating that NDV can induce the host cells to form the YB-1 aggregation,and it located in SG,and SG formation was closely related to YB-1;thus,NDV regulated the host YB-1 in transcription level and translation level.YB-1had an effect on the virus replication.This thesis studied and explained the mechanism at its most basic leve.1 YB-1 gene isolation,identification and genetic evolution analysisIn order to study whether avian YB-1 involved the infectious process of avian RNA virus NDV,AIV,and so on,this study first explored the amplification of YB-1 gene,to be linked to cloning vector and sequenced.We successfully amplified the human and chicken YB-1's mRNA complete open reading frame.Comparing with the mouse of mammal,xenopus of amphibians,fruit fly of insect's YB-1 gene,our results demonstrated that the YB-1 gene homology of human and chicken,mouse,xenopus,fruit fly respectively arrived at 88.3%,96.1%,80.0%,51.0% in nucleotide level,but it arrived at 78.9%,97.8%,85.8%,36.5% in amino acide level.To analyze the difference of human and chicken's gene contributes to realize the difference of YB-1's function in different cells and species andthe reason.Conclusion: The chicken and human's homology at the nucleotide level reached88.3%,and 78.9% at amino acid homology.It suspected that they had the same function.2 The study of NDV effecting on YB-1 subcellular localization and its relationship with SGIn order to study whether YB-1 involved the infectious process of NDV and AIV virus of avian RNA virus,the indirect immunofluorescent assay was first explored the changes of YB-1 subcellular localization after NDV infection.Acording to the latest study report,YB-1 aggregation often correlates with the stress granules(SG).In order to determine YB-1presented puncta aggregation induced by NDV infection that correlated with SG,this study the IFA was explored the subcellular localization of NP protein,YB-1 and TIA of SG's marker in cells infected by NDV;meanwhile,respectively treating cells by ARS and CHX regulated SG formation to study the changes of YB-1 after virus infection.Conclusion:In the normal HeLa cells,YB-1 was evenly distributed dispersion mainly found in the cytoplasm.But YB-1 mainly distributed in the nucleus in chicken's cells.Under NDV infecting HeLa cells,YB-1 did not appear nuclear translocation phenomenon instead of in the cytoplasm producing puncta aggregation.But in infected DF-1 cells by NDV,a lot of YB-1 transfered to the cytoplasm from nucleus.In these two kinds of cells,YB-1 all existed co-localization with SG.But only a little YB-1 and SG co-localized with the NP protein.It suggests that YB-1 was located in SG.In the infected cells by NDV,after SG formation being inhibited,YB-1 aggregation phenomenon also disappeared.3 Study on the regulation of host YB-1 mediated by Newcastle disease virus infectionIn order to study YB-1's puncta aggregation by viral infection correlating with the NDV replication and the molecular mechanism,this study we detected the changes of YB-1's transcription level and protein level after NDV infection via the RT-qPCR,Western Blot,and so on.Subsequently,we screened a pair of siRNA that it effectively inhibited the YB-1,transfecting HeLa cells and utilizing Western blot to detect the expression of NDV NP protein in down-regulated YB-1 cells.In the end,in order to determine whether YB-1bonded with the host and virus mRNA,the fluorescence in situ hybridization(FISH)assay was explored the subcellular localization of YB-1,virus mRNA and host mRNA.Conclusion: YB-1 presented a downward trend in mRNA level and protein level in HeLa cells by NDV infection.In down-regulated YB-1 gene expression HeLa cells,the expression of NDV NP protein level obviously presented increase;but in avian DF-1 cells,the YB-1presented upward tendency in mRNA level with the NDV infection.The result of FISH assay revealed that The YB-1 co-localized with the host mRNA.Instead,it seldom partialy co-localized with viral NP protein' mRNA.