The Molecular Mechanism Of The Effects Of Functional Domains Mutation In The M Protein And The Interaction Of M Protein With Cellular Nucleolar Phosphoprotein B23on 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 approximately15kb 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. In addition to functioning for the assembly and budding of viral particles in the cytoplasm through the co-interaction of M protein with cell membrane, viral nucleocapsid protein and glycoproteins, 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. However, there are still many unknown factors about the functions of M protein in the replication process of NDV. In this study, we characterized the functional domains existed in the NDV M protein and investigated the molecular mechanism underlying the effects of functional domains mutation in the M protein and the interaction of M protein with cellular proteins on the replication of NDV. 1. Characterization of signal sequences determining the nuclear export of the NDV M proteinIn this study, green fluorescent protein (GFP)-labeled assay was firstly used to investigate the subcellular localization of the NDV M protein. The localizations of NDV M protein fused to the C-terminal end of GFP (GFP-M) were examined by fluorescence microscopy. Results showed that the results of GFP-labeled assay were consistent with the localizations of M protein in NDV-infected cells detected by indirect immunofluorescence assay. In addition, eleven candidate nuclear export signal (NES) motifs with striking similarity to the NES sequences reported in other proteins were probed in the M protein of NDV strain F48E8. After transfecting the GFP-M-NESs into CH0-K1cells, three functional NESs were identified when cells were treated with cycloheximide. Then the strength of the nuclear export activity of the three identified M-NESs was evaluated. We found that the nuclear export activity of M-NES1was rated as strong (score4+), whereas M-NES2and M-NES3were rated as weak (score2+). To further confirm the key amino acids determining the nuclear export activity of the M-NESs, the last two hydrophobic residues or all the major hydrophobic residues in the M-NESs were mutated alanine (NES-mt1or NES-mt2). Results revealed that the last two hydrophobic residues, which were conserved among different genotype NDV M proteins, were critical for the export activity of the three M-NESs. Meanwhile, NES-mtl was introduced into the M-NESs to study the effect of M-NESs mutation on the subcellular localization of NDV M protein. We found that single and double NES mutation could not alter the redistribution of the M protein, while only the GFP-M mutant with all three of NESs inactivated was exclusively restricted to the nucleus. In any case, the localization of the M protein was not altered even upon LMB treatment. In conclusion, our results suggested that the nuclear export of the NDV M protein was mediated by three functional NESs through the CRM1-independent nuclear export pathway.2. Mutations in the FPIV motif of Newcastle disease virus M protein attenuate virus replication and reduce virus buddingIn the present study, multiple sequence alignment of paramyxovirus M proteins revealed that a sequence motif FPIV in the NDV M protein was found to be identical to the late domain sequences FPIV identified in the M proteins of parainfluenza virus5and Mump Virus. To generate the recombinant infectious clones harboring alanine (A) single-substitution targeting residues in the FPIV motif, a cDNA clone of NDV strain ZJl (pNDV/ZJ1) was used to introduce individual amino acid substitutions. The FPIV motif mutant viruses were successfully rescued by reverse genetics approaches. And mutation of the residue F or P in the FPIV motif greatly attenuated virus replication and pathogenicity in chicken embryos and avian cells. Then the biological characteristics of these mutant viruses were examined. The MDT results showed a significant increase in the time required by the mutant viruses rZJ1M.F23A (102h) and rZJ1M.P24A (84h) when compared to that required for the parental virus rZJ1(54h). And the ICPI values of rZJl M.F23A (1.64) and rZJ1M.P24A (1.70) also decreased significantly in comparison to that of rZJ1(1.89). In addition, virus budding experiments showed that the FPIV motif sequence within the NDV M protein was critical to NDV virions production, with residues F23and P24playing more important roles than residues125and V26. Together, our results demonstrated that the FPIV motif, especially the residues F and P, within the NDV M protein was essential for the replication and budding of NDV. And the cellular multivesicular body machinery recruited by the FPIV motif was involved in NDV budding.3. A single amino acid mutation, R42A, in the Newcastle disease virus M protein abrogates its nuclear localization and attenuates viral replication and pathogenicityThe NDV M protein is reported to have the highly basic, hydrophobic but not membrane spanning properties. Analysis of the charge distribution in NDV M protein showed that the N-terminal100amino acids are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. This study was undertaken to examine the effects of the basic residues mutation at the N-terminal100amino acids on the subcellular localization of M protein and on the replication and pathogenicity of NDV. Results showed that of all these mutants, only R42A mutation abrogated the nuclear localization of M protein, but did not affect its interaction with viral HN and NP proteins. Multiple sequence alignment of paramyxovirus M proteins showed that this R residue at position42of NDV M protein was conserved in most of paramyxovirus M proteins. To further analyze whether disruption of M protein nuclear localization affects NDV replication and pathogenicity, a cDNA clone of NDV strain ZJ1expressing GFP (pNDV/ZJIGFP) was used to introduce individual amino acid substitution in the M protein. Results of the mutant viruses rescue revealed that the R42A mutant virus grew slowly in chicken eggs. Then the biological characteristics of the parental and mutant viruses were evaluated. The MDT result showed a significant increase in the time required by the M/R42A mutant virus (115h) when compared to that required for rZJ1GFP (52h). And the ICPI and IVPI values of the M/R42A mutant virus also greatly decreased in comparison to the parental virus. In addition, the in vitro growth characteristics and plaque formation of these viruses suggested that R42A mutation reduced the replication and pathogenicity of NDV in avian cells. Meanwhile, the R42A mutant virus caused more mild clinical signs to the birds and had more delayed and much lower virus replication in the chicken organs. In summary, our results indicated that R42A mutation in the M protein attenuated viral replication and pathogenicity caused by the disruption of M’s nuclear localization.4. The interaction of Newcastle disease virus M protein with cellular nucleolar phosphoprotein B23facilitates viral replicationA yeast two-hybrid system was firstly performed to examine the interaction of NDV M protein with cellular nucleolar proteins. The nucleolar phosphoprotein B23was observed to interact with NDV M protein in yeast. And this interaction was subsequently confirmed by GST pull-down assay and, more importantly, by co-immunoprecipitation assay in NDV-infected cells. In addition, we found that NDV M protein accumulated in the nucleolus by binding B23early in infection, but resulted in the redistribution of B23from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids30-60of M and amino acids188-245of B23were required for binding. To further investigate the role B23protein in the subcellular localization of M protein and in the replication of NDV, RNA interference assay was performed to reduce the expression level of B23. Results showed that B23depletion leaded to M accumulation in the nucleus and inhibited NDV replication in DF-1cells. Furthermore, overexpression of B23or M-binding B23fragments affected the normal subcellular localization of M protein in NDV-infected cells and restricted NDV growth. Taken together, our results demonstrated that B23facilitated NDV replication by transferring M to the nucleolus through M-B23interaction. We hypothesized that NDV replication was enhanced by disrupting the structure and function of B23caused by M protein in the nucleolus.