The Molecular Mechanism Of The Effects Of Functional Amino Acids Mutation In The M Protein And The Interaction Of M Protein With Cellular Annexin A6 On The Replication Of Newcastle Disease Virus

Newcastle disease virus(NDV),a member of the genus Avulavirus within the family Paramyxoviridae,is an enveloped virus with a non-segmented,single-stranded,negative-sense RNA genome of approximately 15.1 kb that encodes at least six viral proteins.Of all these structural proteins,the M protein is the most abundant protein in virions and forms an outer protein shell around the nucleocapsid,constituting the bridge between the viral envelope and the nucleocapsid.Like M proteins of other paramyxoviruses,the NDV M protein is demonstrated to be a nucleocytoplasmic shuttling protein.The NDV M protein is observed to localize in the nucleus early in infection and becomes associated with nucleoli and remains in this structure throughout infection.This nuclear-nucleolar localization of NDV M protein is thought to ensure that viral replication and transcription in the cytoplasm proceed smoothly,and also inhibit host RNA and protein synthesis.These studies support the notion that NDV M protein is an essential multifunctional viral protein that plays important roles in the virus life cycle.According to the previous studies on NDV M protein in our laboratory and by others,the M protein plays a critical role in the NDV replication and pathogenicity while the underlining mechanisms are still largely unknown.In this study,we investigated the molecular mechanism of the effects by functional amino acids substitution in NDV M protein and the interaction of M protein with cellular proteins on the replication of NDV.1.A single R36Q mutation in the matrix protein of pigeon paramyxovirus type 1 reduces virus replication and shedding in pigeonsPigeon paramyxoviras type 1(PPMV-1)is an avian pathogen capable of specifically infecting pigeons and is often considered an antigenic and host-adapted variant of avian paramyxovirus type 1(APMV-1,NDV)in pigeons.It has been reported that this host-adaption of PPMV-1 is related to the viral genes and proteins.However,up to now,how this host-specific adaption of PPMV-1 is related to its biological characteristics is unknown.The M protein,which is usually considered the third NDV envelope protein following the HN and F proteins,plays a crucial role in the viral replication cycle.Additionally,it has been suggested that M protein plays a key role in the molecular evolution and phylogenetic relationships of PPMV-1.In the present study,in order to find the key unique amino acid responsible for the host specificity of PPMV-1,145 entire genome sequences of APMV-1 were obtained from GenBank database and aligned for analysis.There were one,one,one,and four unique amino acid residues in the M,F,HN,and L proteins of PPMV-1,respectively.Furthermore,the results revealed that R or K(37/2)is a unique amino acid at position 36 of M.At the same time,the R36 was detected to be a positive-selected site based on previous study.To investigate the role of R36 in PPMV-1,the cDNA clone pNDV/071204 of PPMV-1 strain Js/07/04/Pi(071204)was used to generate a mutation R36Q.For comparison,the full-length cDNA clone pNDV/ZJ1GFP of NDV ZJ1 strain expressing green fluorescent protein was used to construct the Q36R mutation in the M protein,recombinant viruses were named r071204-M.R36Q and rZJ1GFP-M.Q36R.The results showed that the R36Q mutation significantly attenuates pathogenicity in chickens,viral growth in both chicken embryo fibroblasts(CEFs)and pigeon embryo fibroblasts(PEFs),virus replication,in comparison with the wild-type virus.In addition,the pigeon challenge experiment showed that the virus with R36Q mutation(r071204-M.R36Q)caused shorter and lower virus shedding when compared with those of the parental virus(071204).However,the Q36R mutation had no significant effect on biological characteristics of ZJ1 in vitro and in vivo.These results suggest that R36 is a key residue that evolved during the adaptation of PPMV-1 in pigeons.2.Simultaneous mutation of G275A and P276A in the matrix protein of Newcastle disease virus decreases virus replication and buddingIt is reported that in the late viral infection the M protein needs to be recruited on the inner surface of the host cell membrane to to facilitate the viral particles assembly and budding,and this process depends on a specific fold with two α-helix wrapped two antiparallel β-fold which interacts with the membrane.In measles virus and Sendai virus,the paired glycine(G)near the C-terminus of the M protein may form the specific turn.Similar amino acids(glycine-proline[GP],at position 275-276)exist in the M protein of NDV.However,the role of these residues in the replication and pathogenicity of NDV is unknown.In this study,the results of previous studies were firstly verified.Multiple sequence alignment of NDV M proteins with other paramyxovirus M proteins revealed that a LGP motif in the NDV M protein was found to be identical to the paired G identified in the M proteins of other paramyxoviruses.The 3D structural simulation analysis showed that the GP/AA mutation influence the fold angle may affect the M protein function.And then,two recombinant NDVs with the sequence GP/AA or LGP/GGL in the M protein were generated to investigate the role of this conserved sequence.The results showed that GP/AA mutation significantly reduced the recombinant virus replication and pathogenicity in chicken embryos and DF1 cells.Moreover,the animal infection experiment also demonstrated that the GP/AA mutation attenuated virus replication and pathogenicity in chickens.In addition,budding experiments on the mutant viruses revealed that the GP/AA mutation reduced virus budding and virus replication in DF-1 cells,which also explains why the virus replication capacity decreased.Together,our results suggest that the GP sequence plays a critical role in the life cycle of the virus.However,further study by structural proteomics is needed to elucidate the detailed mechanism of GP sequence affecting viral budding.3.The functional domain in C-terminus of M protein of Newcastle disease virus affects the early viral replicationIn this study,mutations of amino acids that affect the dimer array formation in the C-terminus of M protein of the NDV ZJ1 strain were performed and the effect of point mutations on the subcellular localization of NDV M protein were observed.Subsequently,the recombinant viruses with different mutations were rescued by reverse genetics to investigate the effect of mutations in viral replication and pathogenicity.The results showed that the single-point mutation of D255,E258,R262,R263 and 255/258(DE)double-point mutation in the M C-terminal of M protein did not affect the nucleus and nucleolus localization of M protein,however,the double-point mutations of 262/263(RR),255/263,258/262 resulted in the localization of M protein from nucleus and nucleolus to cytoplasm.And then,the full length cDNA clone pNDV/ZJ1GFP of NDV ZJ1 strain which expresses green fluorescent protein were used to construct full-length plasmid pNDV/ZJ1GFP-Mwt with single-point mutation or double-point mutation in the M protein,and eight recombinant viruses were successfully rescued by reverse genetics technology.Then the biological characteristics of the parental and mutant viruses were evaluated.The results showed that both single-point and double-point mutations could not markedly change the biological characteristics of these viruses.However,the double-mutation recombinant viruses 255/258(DE),262/263(RR),255/263,258/262 in DF1 cells showed a significant decrease in viral proliferative capacity,CPE formation ability and GFP expression in the early stage of viral replication.It is indicated that the double-mutations in the C-terminus of M protein could reduce the viral replication and pathogenicity during the early stage of NDV replication.In summary,our results demonstrated that the amino acids that influence the M protein dimer array formation could affect the nuclear localization of NDV M protein and thus attenuate the NDV early replication and pathogenicity in DF1 cells.4.The Annexin A6 interacts with Newcastle disease virus matrix protein and negatively regulates viral replicationDue to the limited capacity of the virus encoded proteins,the virus could not encode all of the proteins that required for replication,thus,the host cell proteins or cell structure components are often needed to complete the virus replication process.In this study,a laser confocal microscopy was firstly performed to examine the localization of NDV M protein with cellular annexin A6(ANXA6)protein in cells.The cellular ANXA6 protein was observed to co-localize with NDV M protein in the cytoplasm.And then,GST pull-down assay and,more importantly,co-immunoprecipitation assay in NDV-infected cells were used to further confirm that NDV M protein could interact with the cellular ANXA6 protein.In addition,by the His Pull-down method,in vitro binding studies utilizing deletion mutants indicated that amino acids 250-280 of M was required for M-ANXA6 binding.To further investigate the role of the M-ANXA6 interaction in the replication of NDV,RNA interference assay and overexpression ANXA6 in host cells were performed.The results showed that inhibition of ANXA6 protein expression could promote the production of virus in infected cells and thus enhance the viral replication,indicating that the ANXA6 protein could inhibit the NDV replication.On the other hand,intracellular overexpression of the ANXA6 protein significantly reduced the CPE induced by NDV-infected cells and inhibited the proliferation of NDV viral,indicating that the overexpression of the ANXA6 protein in the host cells significantly reduced NDV replication.Taken together,our results demonstrate that the cellular ANXA6 protein could negatively regulate NDV replication through M-ANXA6 interaction.