The Molecular Mechanism Of Newcastle Disease Virus V Protein Mediates IRF7 To Antagonize IFN-β

Newcastle disease virus(NDV)is highly pathogenic and strongly infectious.Office international desépizooties(OIE)has listed ND as a legal-reported disease,allowing that countries and regions could establish reasonable trade restriction to control NDV transmission.Although scientist have done a great deal of research on ND prevention and control methods,and no efficient universal solutions has been formed;repeated ND outbreak and emerging NDV new genotype also indicating that regular control approach is yet to be developed.To control ND at the source,we should have a thorough understanding the pathogenic and transmission mechanism of NDV.In this paper,we studied and analyzed the mechanism of how V protein regulates host innate immune response in viral pathogenic process,providing theoretic basis and experiments data for ND control.Our works involve three parts:The first part is the preparation of V-protein-specific antibody.Specific antibody is important tool to mark time and space distribution of viral protein in infected cells,while no commercial anti-V protein is available.Experiments were carried out to detect and evaluate expression of V protein in NDV infection:prokaryotic recombinant proteins were purified and used as antigen to prepare poly-clone antibody Anti-V_C,which can bind V protein specifically,and Anti-V_Nand Anti-P,which can detect both protein P and V.Assays showed that all three kinds of antibodies could used for detection of NDV-infected samples,laid the foundation for continued study of NDV V protein mediated transportation factors regulation.The second part is about to study the relationship between V protein structure,transportation factor IRF7 and type-I interferons.Previous studies have proved that NDV can induce various interferons,initiate anti-viral response in infection while V protein have a function to reduce the IFN response.In this paper,different dosages of NDV and poly(I:C)(to mimic ds RNA)both could induce type-I interferons response and IRF7/IFN-βresponse is performed as the primary response models.poly(I:C)stimulation and NDV infection shared a similar but earlier IRF7/IFN-βresponse trend,and IRF7 always reached its peak earlier than IFN-βin both models.To verify the regulate function of IRF7 to IFN-β,ch-IRF7 was cloned and overexpressed in DF-1 cells,and we find IFN-βresponse is enhanced positively correlation with ch-IRF7 protein level.We also confirmed ch-IRF7 as a positive signal in innate anti-viral response because its overexpression significantly decreased NDV multiplication.To further study the structural basis and regulation model of V protein interfering IFN response,V protein was segmented expressed as two domains,then the effect of both V protein and domains to IRF7 gene transcription activity,IFN-βsecretion and virus multiplication was detected.results showed NDV V protein could down-regulate IRF7 gene transcription-activity and IFN-βsecretion and increase virus multiplication;the C-teminal domain of V protein could inhibit IRF7 but could not affect IFN-βsecretion and virus multiplication.Results above indicated that C-terminal structure of V protein is essential to inhibit IRF7,while the function to antagonize the IFN-βresponse and enhance virus multiplication rely on complete structure.Further study on how V protein regulating IRF7,we found that V protein interacts with ch-IRF7 and its upstream molecule ch-TRAF6.Additionally,V protein reduce protein level of ch-IRF7 but not regulate ch-IRF7 nucleus transportation.The third part is to identify and screen V protein binding host proteins in DF-1 cells.Classical studies mainly focus on V protein functions on anti-innate immune system.Recent reports have found that non-structural proteins of many virus are involved in various cell processes such as cell-cycle,protein translation and energy metabolism thus regulate virus replication,but system studies to find out which proteins V protein could bind in chicken cells have not performed.This paper expressed two kinds tag-fusion V proteins,and co-IP assays rely on anti-Flag and anti-HA magnetic beads were applied combined with MS-MS method to screen V protein natural binding host proteins.Identified proteins were aligned in uniprot database.Two tag-antibodies identified 195 and157 proteins,respectively.Gene annotation shows:V-binding proteins have various catalytic activities,indicating that V protein may own several sites for post-translation modification;the nucleus-location of V-binding proteins suggesting that V protein can transport between cytoplasm and nucleus;pathway annotation reveals that V-binding proteins are involved in a variety of signal pathways mainly related to cell cycle and metabolism.Further screening by the union of two proteins sets obtained 14 proteins with both tags positively and these proteins have high reliability,giving new directions for research of V protein functions.Genotype Ⅶ NDV strain was taken as the study object in this study,we analyzed the relationship of viral V protein structure,transcription factor IRF7 and type-I interferons antagonism.Our results enrich the molecular mechanism of NDV pathogenicity,provide theory basis to construct new-type NDV vaccine by modifying NDV gene,and give references to regulate host immune response clinically and control ND.Additionally,we screen 14 V-binding proteins in DF-1 cells,providing starting points for system study of V protein functions.