Subcellular Localization Analysis Of Porcine Epidemic Diarrhea Virus N Protein

Porcine epidemic diarrhea(PED) caused by porcine epidemic diarrhea virus(PEDV), is an acute and highly contagious enteric disease in swine. The disease is characterized by severe enteritis, vomiting and watery diarrhea. Its prevailence has resulted in heavy economic losses in swine industry. However, the research about the pathogenic mechanism of PEDV is unclear. PEDV N protein is a multifunctional phosphorylated protein composed of 441 amino acids(aa),which exhibited high basic and serine content. It is highly basic enabling it to interact with genomic RNA to form the structure of the viral nucleocapsid.The N protein is the most abundant virus-derived protein produced throughout infection. Research indicated it plays an important role in the induction of immunization and the pathogenic mechanism of viral infection. So, In this study, subcellular localization properties of porcine epidemic diarrhea virus nucleocapsid protein were carried out. This will provide some reliable evidence for the elucidation of the pathogenic mechanism of PEDV.In this study, the N protein showed high soelectric point, two potential nuclear(or nucleolus) localization signals ((N (or No) LS) were characteristiced by bioinformatics analysis, and are basic amino acids riched region. These signals are located in hydrophilic regions, are accessible.The ORF of N gene was cloned and subcloned into pET30a (+), and expressed in E.coli fused with 6his in frame by IPTG induction. The 6His-tagged fusion protein was purified and used to immunize female BaLB/c to generate antiserum against PEDV CV777 N protein. To identify the localization of N protein in PEDV CV777 infected Vero E6 cells at MOI=1 where also other viral proteins were expressed, cells were fixed at 24 h post-infection and analyzed by indirect immunofluorescence assay using the antiserum. The results showed the N protein localized merely to the cytoplasm in most infected cells, there also a few cells showed that the N protein localized predominantly to the cytoplasm whilst in a few cells a weak fluorescence label was also observed in the nucleus under confocal microscopy.The subcellular localization of N gene was investigated. The recombinant plasmid pcDNA3.1(+)-N was transfected into Vero E6,then the cells were observed under confocal microscopy. The flow cytometer was used to analysis the cell cycle distribution after N protein expression. These results indicated that the transfected cell showed the similar localization properties as the infected cells, and Vero E6 cells expressing N protein exhibited cell growth retardation most likely through G2/M arrest. These indicated that N protein may inhibited the synthesis of cell RNA and protein in G2 / M phase to facilitate virus propagation.Based on amino acid sequence comparisons, coronavirus N proteins can be separated into three conserved structural domains. So the plasmid about truncated different regions of N gene and predicted localization signals contained AcGFP1-tagged was transfected into Vero E6 cells, which not destroyed the integrity of the signals. And the DsRed-B23.1 was used to sign nucleolus.The exhibition of N protein nucleolar localization and co-localization characteristics in co-transfected cell by confocal microscopy observation.The live imaging indicated that AcGFP1 and DsRed uniformly localized to both the cytoplasm and nucleus; whereas AcGFP-N localized to both cytoplasm and nucleolus, and also showed co-localization with nuclear phosphoprotein B23.1. AcGFP-NRâ… mainly localized to nucleolus; AcGFP - NRâ…¡mainly localized to the nucleus and nucleolus, but also the distribution of protein in nucleolus larger than that in the nucleus; AcGFP-NRâ…¢localized to the cytoplasm and nucleus, no nucleolar localization; AcGFP-pat7 mainly localized to nucleus; AcGFP-patx showed strong nuclear and nucleolar localization. The deletion mutagenesis analysis indicated the localization characteristics about AcGFP-NRâ…¡â–²Pat7and AcGFP-Nâ–²Patx is the same as AcGFP - NRâ…¡and AcGFP-N. These results indicated NRâ… protein and NRâ…¡protein certainly contained nucleolar localization signals which can not be predicted through bioinformatics,that provided reliable data for futher functional nucleolar localization signals identification.The B23.1 gene was cloned into pGEX-6p-1 vector to express the protein. The result of SDS-PAGE assay showed that the protein was largely expressed,The GST-tagged protein was purified. The western blot analysis showed that the target protein has a good reactivity. It is an important foundation to analysis protein interaction between N protein and nucleolar protein B23.1 in future.