Nucleocytoplasmic Shuttle Mechanism Of Newcastle Disease Virus M Protein And Its Effect On Virus Proliferation

Newcastle disease viruses(NDV)are among the pathogens of highly contagious diseases that afflict the poultry industry worldwide.NDV belongs to subfamily paramyxoviridae and the genus Avulavirinae,Orthoavulavirus,which is also known as avian paramyxovirus type ?.NDV is a kind of enveloped virus.Its genome is composed of single-stranded negative stranded RNA,which consecutively encodes NP,P,M,F,HN and L six structural proteins in sequence.Among them,the gene length of the Matrix(M)protein is about 1.2 Kb.The relative molecular weight is about 40 k Da.The gene sequence of M is relatively conservative among different NDV strains.M protein is an important structural protein encoded by paramyxoviridae,which forms a grid array on the inner surface of viral capsule.Depends on itself nuclear export signal(NES)and nuclear localization signal(NLS),M protein has the characteristics of Nuclear cytoplasmic trafficking.By interacting with surface glycoproteins and RNP complexes,it acts as a bridge between RNP complexes and glycoproteins to drive viral assembly and budding and concentrates them on the plasma membrane of the host cell.Compared with other paramyxoviridae members such as NIV,RSV,and MUV,little is known about the role of nuclear-cytoplasmic trafficking of NDV M protein in viral replication.To clarify the function of NDV M protein and the effect of its " nuclear-cytoplasmic trafficking " characteristic on viral replication,in this study,we regard the M protein of velogenic NDV Herts/33 strain and lentogenic vaccine strain La Sota as the study of the object.Based on a series of experimental techniques such as indirect immunofluorescence(IFA),Western blotting(WB),transmission electron microscopy(TEM),and co-immunoprecipitation(CO-IP),we conducted a series of experiments and obtained the following results:Firstly,the efficiency of “nuclear-cytoplasmic trafficking” of NDV M protein differed between velogenic and lentogenic strains.The phenomenon of nuclear-cytoplasmic trafficking existed in both infection and transfection states,and virus-like particles could be produced when NDV M protein was expressed individually.By IFA,WB,and EM methods,we observed that the phenomenon of nuclear-cytoplasmic trafficking existed in both infection and transfection states,and virus-like particles could be produced when NDVM protein was expressed individually.Meanwhile,the efficiency of “nuclear-cytoplasmic trafficking” of NDV M protein differed between velogenic and lentogenic strains that is the efficiency of “nuclearcytoplasmic trafficking” NDV Herts/33 M protein is higher than that of La Sota M protein.Furthermore,this difference will lead to the different efficiency of viral budding of VLP driven by M protein,that is,the efficiency of Herts/33 M protein to produce VLP is higher than that of La Sota M protein.Secondly,amino acids at positions 247 and 263 jointly determine the efficiency difference of nucleation and budding of NDV M protein.To explore the specific mechanism of the different efficiency in “nuclear-cytoplasmic trafficking” of M protein between velogenic and lentogenic NDV strains,point mutations were performed on the 247-position and 263-position amino acids on NLS of both M proteins.Using the IFA,we observed that K247 R,R263S,K247R&R263S point mutations of Herts/33 M protein all changed the nuclear localization of M protein to different degrees,and reduced the nuclear export efficiency of M protein.However,the point mutations of R247 K,S263R,R247K&S263R of La Sota M protein all improved the nuclear nucleation efficiency of M protein to different degrees.Additionally,the supernatant of cells transfected with M protein was collected for centrifugation concentration,and the production of VLP was detected and compared by WB.We found that the Herts/33 M protein point mutation resulted in a decrease in the production of VLP,while the point mutation of La Sota M protein increased the production of VLP.The above experiments preliminarily showed that the difference in the “nuclearcytoplasmic trafficking” of M protein between velogenic and lentogenic NDV strains was caused by the amino acid difference in nucleoplasmic localization signal,and the difference in nuclear export efficiency of M protein would also lead to the difference in VLP release efficiency.Thirdly,the nuclear export of M protein is positively correlated with virus yield,and the nuclear exportability of M protein promotes the viral proliferation of NDV.Using NDV reverse genetic platform,we constructed the full-length plasmid of La Sota M protein point mutation with TVT-La Sota C5 plasmid as skeleton and successfully rescued four recombinant viruses,namely r La Sota-WT,r La Sota-R247 K,r La Sota-S263 R,and r La Sota-R247K&S263R.The results of biological characteristics showed that the titer of haemagglutination assay(HA)of the recombinant virus was higher than that of the parent virus strain,and the MDT(115h)was significantly shorter than that of the parent virus strain(142h),but the ICPI value was not different.Our results indicate that M protein R247 K and S263 R mutations can improve viral replication by affecting the nuclear localization of the protein.Fourthly,the M protein of the velogenic Herts/33 strain has more complex ubiquitination modification,while the M protein of the lentogenic La Sota strain has more simple ubiquitination modification.The results of Co-IP identified that NDV Herts/33 M protein and La Sota M protein were significantly different in ubiquitination modification: Herts/33 M protein had more complex ubiquitination modification,while Lasota M protein ubiquitination modification was more single.Fifth,the interaction sites between NDV M protein and ubiquitin are mainly in the nucleus and its surroundings,but there are a large number of M proteins that have not been ubiquitinated in the nucleus.The results of BIFC confirmed that the interaction site between NDV M protein and ubiquitin is mainly in the nucleus and its surroundings,but there are a large number of M proteins that have not been ubiquitinated in the nucleus,so we speculated that the ubiquitination of M protein may occur in the nucleus,and M protein needs to be ubiquitinated in the nucleus before it can function.At the same time,we fused UB-WT/UB-K0 into the terminal of La Sota and its mutants to simulate mono-ubiquitination and found that both forms could lead to nuclear export,indicating that the mono-ubiquitination of NDV M protein was sufficient to meet the need of nuclear export.Sixth,the K247 amino acid site of NDV M protein is the key ubiquitination site.After comparing the M protein structures of other paramyxoviruses,we found that the amino acid at K247 of NDV M protein was located in the ubiquitination domain similar to that of other paramyxoviruses,so it was speculated that K247 was the ubiquitination site.Through co-expression of Flag-M and HAubiquitin-K0 in 293 T cells,flag coupled magnetic beads were used for immunoprecipitation of M protein,and HA labeled antibody was used for WB detection.It was found that ubiquitination bands increased in R247 K and decreased in K247 R.The K247 amino acid of M protein was preliminarily identified to be ubiquitinated,and there was a certain correlation between ubiquitination and “nuclear-cytoplasmic trafficking” and viral budding.Finally,by simulating the structure of NDV M protein,we found that NDV M has a domain similar to that of other paramyxoviruses.Amino acid 263 appears at the interface of M protein dimer interaction,while amino acid 247 is spatially close to the plasma membrane,which is located in the same domain as the ubiquitination site of other paramyxoviruses M proteins.In summary,we regard the “nuclear-cytoplasmic trafficking” of M protein as the pointcut of the study,and we found that there were strain differences in the efficiency of “nuclear-cytoplasmic trafficking”and the ubiquitination modification level of M protein of velogenic and lentogenic NDV strains.At the same time,the unique collaborative signal peptide of NDV M protein makes use of the ubiquitin to influence virus replication by influencing the efficiency of “nuclear-cytoplasmic trafficking”.In this study,we clarified the behind mechanism and biological significance of the efficiency difference of the “nuclearcytoplasmic trafficking” between velogenic and lentogenic NDV strains.All those results lay a foundation to further explore the significance of “nuclear-cytoplasmic trafficking” for virus replication and viral budding,which will bring positive significance to the modification of La Sota vaccine strain.