Study On The Role Of Chicken Matrix 3 Protein In The Replication Of Newcastle Disease Virus

Newcastle disease virus?NDV?is an enveloped virus with a non-segmented,single-stranded,negative-sense RNA genome,which encodes at least six viral structural proteins in the order of 3'-NP-P-M-F-HN-L-5'.The matrix protein?M?is a 40 kD hydrophobic protein encoded by the NDV M gene and is inside the viral envelope.Like most paramyxoviruses M proteins,the NDV M protein is demonstrated to be a nucleocytoplasmic shuttling protein,showing that M protein interacts with importin?1 through its own nuclear localization signal?NLS?and enters the nucleus in a RanGTP-dependent manner early in infection,and then enters the cytoplasm via a CRM1-independent manner through three nuclear export signals in the M protein later in infection.Matrin 3?MATR3?,mainly localized in the nucleus,is one of the first 12 major nuclear matrix proteins and is widely expressed and highly conserved in different cell types.MATR3 is reported to participate in the gene transcription regulation,the splicing and stability of pre-mRNA,DNA damage repair and cell proliferation.In our previous studies,the MATR3 is found to interact with NDV M protein by yeast two-hybrid system from the cDNA of chicken embryo fibroblasts,but the role of this interaction in NDV replication is still unclear.In this study,the chicken MATR3 gene was cloned and analyzed by bioinformatics,and the NLS of MATR3 was identified.The recombinant plasmids expressing NDV M protein and MATR3 were constructed,and fluorescent co-localization and Co-IP assay was used to verify the interaction between M protein and MATR3.Finally,RNA interference technique was used to interfere with the expression of MATR3 in BSR-T7/5 cells to study its role in NDV replication.1.Cloning and bioinformatics analysis of chicken MATR3 geneSpecific primers were designed and used to amplify of chicken MATR3 gene from DF-1cells.The physicochemical properties,secondary structure,tertiary structure,functional domain and phylogenetic tree of chicken MATR3 were predicted by the bioinformatic tools.[Results]The full-length ORF sequence of chicken MATR3 gene was 2 709 bp and encoded902 amino acids.The molecular formula of chicken MATR3 was C₄₃₆₂H₆₉₀₃N₁₂₆₇O₁₄₁₈S₂₉9 with a relative molecular mass about 100.71 kDa and pI 5.79.Protein secondary structure prediction showed that chicken MATR3 contained abundant secondary structure and were mainly random coil?47.67%?and alpha helix?33.81%?.Functional domain prediction revealed that the protein had two zinc finger domains and two RNA recognition motifs.The results of gene homology and phylogenetic tree revealed that chicken MATR3 gene had a close relationship with turkey.2.Prediction and identification of nuclear localization signal of Chicken MATR3The NLS in chicken MATR3 was predicted by the online software.Overlap PCR primers were designed to recombinant plasmids of chicken MATR3 with the deletants were constructed and then transfected into cells.The subcellular localization of the deletants was observed and analyzed to determine the NLS of chicken MATR3.The recombinant plasmids expressing the deletion of basic amino acids close to NLS were also constructed to examine their roles in the nuclear localization of chicken MATR3.The results showed that there were four putative NLS?pNLS?existed in chicken MATR3,and the deletion of pNLS2^{?596?PSDKKSK602}disrupted the nuclear localization of chicken MATR3.In addition,the adjacent basic amino acids of NLS did not participate in the NLS-mediated nuclear localization of chicken MATR3.Moreover,we also found that the NLS motif in MATR3 were conserved among different species,and also had the same nuclear import ability as the NLS of SV40 large T antigen.3.Verification of the interaction between chicken MATR3 and NDV M proteinSpecific primers were designed and used to construct recombinant eukaryotic expression vector pDsRed-MATR3,pCMV-Myc-MATR3 and pEGFP-M.The plasmids pDsRed-MATR3and pEGFP-M were co-transfected into cells to observe the co-localization of chicken MATR3 and M protein.Then the plasmids pCMV-Myc-MATR3 and pEGFP-M were co-transfected into cells and Co-IP was used to identify the interaction between chicken MATR3 and M protein.The results showed that EGFP-M and DsRed-MATR3 fusion proteins were co-located in the nucleus The results of Co-IP showed that EGFP-M was co-precipitated with Myc-MATR3 and Myc-MATR3 was also co-precipitated with EGFP-M.These results demonstrated that chicken MATR3 could interact with NDV M protein in the nucleus.4.Effect of MATR3 interference expression on NDV replication in cellsMATR3 siRNA was transfected into BSR-T7/5 cells to verify the effect of MATR3siRNA on the expression of exougeous MATR3 in cells.And then 1 MOI of NDV was used to infect cells after the MATR3 siRNA transfection at 36 h.The cytopathic effects and the virus titer of the supernatant at different time points were examined.Meanwhile,the mRNA expression levels of NDV M gene in cell pellet and cell supernatant after virus-infected cells at 24 h was detected by quatative real-time PCR.The results showed that significant cytopathic effect appeared after virus-infected cells at 24 h,and the virus titer reached the maximum at 36 h;the cytopathic and virus titers of the MATR3 siRNA group were lower than that of the Control siRNA group and the Normal group.In addition,the mRNA expression level of NDV M gene in MATR3 siRNA group was also lower than that in Control siRNA group and Normal group.The above results indicated that interfering expression of MATR3 in cells could inhibit NDV replication.