Nuclear Localization And Function Of Newcastle Disease Virus V Protein

Newcastle disease(ND),caused by Newcastle disease virus(NDV),is an acute,septic and highly contagious avain disease,causing huge economic losses to the poultry industry.As an important non-structural protein of NDV,V protein encoded during virus replication,has a variety of biological functions.The function of the viral protein is not only closely related to its structure but also related to the subcellular localization of the viral protein.At present,there are few studies on the subcellular localization of V protein,and its structural basis and biological significance need to be further clarified.In this study,the subcellular distribution of V protein was investigated through nuclear-plasma separation,Western Blot and laser scanning confocal microscope,combined with gene deletion and site-directed mutagensis to analyze its structural basis.Besides,the effect of nuclear localization of V protein on its function was explored initially.The main researchs are as follows:1.Study on the subcellular localization of V proteinIn order to determine the subcellular localization of V protein,we first examined the subcellular distribution of r GM in infected DF-1 and CEF cells,and the r GM is belong to genotype Ⅶ.The results showed that only a small amount of V protein was detected in the cytoplasm after 6 hpi,and it was clearly visible in the nucleus after 12 hpi.The subcellular distribution of V protein in DF-1 cells infected with NDV of different genotypes showed that V protein could enter the nucleus after the DF-1 cells were infected by E105 and F48E9,the others were distributed in the cytoplasm.In addition,the subcellular distribution of the eukaryotic expression plasmid of N1F-V in DF-1 cells demonstrated that V protein mainly distributed in the cytoplasm,and a small amount was localized to the nucleus.2.Identification of V protein nuclear location related domain and key amino acid sitesAfter confirming that the V protein can enter the nucleus,three possible nuclear localization signals(NLS)were predicted and the mutants of V protein were constructed through deleting the predicted nuclear localization sequences.NLS-2(aa138-167)or NLS-3(aa171-200)were identified as functional domains of V protein entring into the nucleus.Since the alkaline amino acid in NLS play a crucial role in the process of protein entring into the nucleus,the alkaline amino acids in NLS were mutated to alanine by site-directed mutagenesis.Then the nuclear distribution of the mutants were detected,the findings indicate that R142 and R152 sites could affect the nuclear localization of V protein.3.Effect of V protein nuclear localization on its functionInterferon and apoptosis play an important role in virus infection and pathogenicity.Extensive researchs have focused on the mechanism of V protein antagonizing type I interferon and regulating apoptosis.However,it is unknown whether these functions are related to the nuclear localization of V protein.Therefore,in this study,the effect of nuclear localization of V protein on the transcription of IFN-β was explored by dual luciferase reporter gene and q-PCR.The results showed that the deletion of NLS-2 or NLS-3 resulted in the loss of IFN-β transcriptional activity.Although the mutation of R142 and R152 amino acids can still inhibit the production of IFN-β,the inhibition is weaker compared to the wild-type V protein.IRF7 is an important upstream molecule that regulates the production of interferon,the expression of V protein can down-regulate the protein level of IRF7.In order to clarify whether the nuclear location of V protein can inhibit the expression of IRF7,we examined the effect of V protein on the expression of IRF7 after co-transfection.The results showed that the deletion of NLS-2 and NLS-3 or mutations at R142 and R152 sites had no effect on the expression of IRF7,suggesting that nuclear localization of NDV V protein participates in inhibition of IFN-β through IRF7degradation-independent mechanism.To investigate the effect of nuclear localization of V protein on apoptosis,flow cytometry was used to detect the apoptosis rate of DF-1 cells induced by N1F-V,V-ΔNLS2,V-ΔNLS3 and V-R142152 A.The results showed that the V protein of deleting NLS-2 and NLS-3 induced apoptosis of DF-1 cells were 3.86% and 7.17%,respectively;The apoptotic rate of DF-1 cells induced by mutation of R142 and R152 amino acid sites of V protein was6.03%,all of them were significantly higher than the 2.32% apoptosis rate of wild-type V protein.It is suggested that the nuclear localization of V protein has a positive effect on inhibiting apoptosis of DF-1 cells.Taken together,our study is the first to confirm that NDV V protein can enter the nucleus,defined two regions of V protein(aa138-167 and aa171-200)and important amino acid sites(R142 and R152)are responsible for nuclear localization.In addition,we found that the nuclear localization of V protein is involved in its inhibitory effect on IFN-βproduction and cell apoptosis.These findings elucidated the relationship between the nuclear location and function of V protein and may provide a new perspective for understanding innate immune of virus resistance.