Mechanistic Study Of NP Protein Interacting With Host To Regulate Newcastle Diseas Virus Replication

Newcastle Disease(ND),an acute and severe infectious disease,was caused by Newcastle Disease Virus(NDV).NDV seriously threatens the development of poultry industry and has been listed as a disease that must be reported to OIE.NDV is a single-stranded negative band RNA virus with encapsulated membrane.Its genome can encode six structural proteins and two non-structural proteins.Among them,nucleocapsid protein(NP)is the most abundant structural protein.NP protein forms ribonucleoprotein complex(RNP)with phosphorus(P),large protein(L)and viral nucleic acid,which is the basic unit for viral replication.NP protein also has other functions,such as mediating cellular immune response,influencing virus virulence and inducing autophagy.The information indicates that NP protein is a multifunctional protein.However,there is no relevant report what kinds of host proteins relate to function of NP protein.In order to reveal the molecular mechanism of NP protein functions,this research mainly studies from the following four parts:(1)In order to verify the effect of NP protein on NDV replication,the eukaryotic expression vector of NP protein was constructed and its normal expression in DF-1 cells was verified.Then the NP protein was overexpressed in DF-1 cells,and the virus content was detected by Q-PCR,plaque and Western blot.The results showed that the overexpressed NP in DF-1 cells could promote the virus replication.In order to further study the mechanism of NP protein promoting virus replication,the expressions of IFN?,IFN? nd IFN? were detected by Q-PCR.The results indicated that NP protein may promote virus replication by inhibiting interferon production.(2)To explore the host factors that NP protein targeted to,the NP gene was cloned onto the bait plasmid pGBKT7 of yeast two-hybrid.After being verified by self-activation and qualified by toxicity detection,pGBKT7-NP was used as the bait to screen the cDNA yeast library of chicken embryo fibroblasts,and a total of 20 host proteins including,PIAS4(Protein inhibitor of activated STAT4)and PSMA7(Proteasome subunit alpha type 7)were finally obtained.(3)In order to explore the relationship between PIAS4 and NP protein,NP protein was overexpressed in DF-1 cells,and it was found that NP protein could inhibit the expression of endogenous PIAS4 by Q-PCR detection.Then,PIAS4 was overexpressed in DF-1 cells and endogenous PIAS4 was knocked down with siRNA,and the virus content was detected by Q-PCR,plaque and Western blot.The results showed that PIAS4 could inhibit virus replication in DF-1 cells.Moreover,overexpression of NP protein in DF-1 cells inhibited endogenous PIAS4 expression.In order to further study the antiviral mechanism of PIAS4,VSV infection experiment and Q-PCR were performed to detect the content of cell supernatant or interferon in cells after overexpression of PIAS4,and the results indicated that PIAS4 may inhibit virus replication by promoting the expression of interferon(IFN).(4)In order to verify the function of PSMA7,the eukaryotic expression vector of PSMA7 protein was constructed and its normal expression in DF-1 cells was verified.Then PSMA7 was overexpressed in DF-1 cells and endogenous PSMA7 was knocked down by siRNA,and the virus content was detected by Q-PCR,plaque and Western blot.The results showed that PSMA7 could inhibit virus replication in DF-1 cells.Moreover,overexpression of NP protein in DF-1 cells inhibited endogenous PSMA7 expression.In order to further study the antiviral mechanism of PSMA7,VSV infection experiment and Q-PCR were performed detect the content of cell supernatant or interferon in cells after overexpression of PSMA7,and the results indicated that PSMA7 may not play an antiviral function through interferon signaling pathway.In conclusion,NDV NP protein can interact with PIAS4 and PSMA7,and inhibit the expression of the two proteins to promote viral replication.