Mechanism Investigation For Nuclear Localization Signal Mediating Porcine Circovirus Type 2 Cap Protein Nuclear Export

Nuclear localization signal(NLS)is a peptide,which consists of either one or two stretches of basic amino acids and is necessary for nuclear protein import.Studies have shown that NLS exists widely in the biology field and mediates proteins or nucleotide cytoplasmic trafficking to complete modification,folding or interaction with other proteins.PCV2 Cap protein is a major structural protein to form the viral capsid protein that surrounds the single-strand genomic DNA.A special arginine-rich N-terminus(1-41aa)of Cap protein is described as a nuclear localization signal to accumulate Cap protein into nucleus.However,mechanism for the Cap protein nuclear export remains unclear.To this end,studies were carried out to investigate the mechanism of Cap protein nuclear export as following: 1.PCV2 d Cap protein self assembles into virus-like particle in insect cells.A PCV2 d Cap gene was cloned and sequenced for bioinformatic analysis.Four domains were found to be different with those Cap proteins from representative PCV2 strains: PCV2 a,PCV2b and PCV2 vaccine strains.The PCV2 d Cap gene was expressed via Bac to Bac expression system in insect cells,and was identified by Western blot,IFA,MS,EM and Immuno-EM methods.Results have shown that PCV2 d Cap protein was highly expressed in insect cells and recognized by the PCV2 positive serum through IFA assay.Furtherly,the PCV2 d Cap protein self assembled into virus-like confirmed by EM and Immuno-EM assay.2.One hybridoma to recognize the PCV2 VLP was prepared.Two forms of Cap protein were expressed and purified from insect cells.Cap protein with the NLS(Cap),was used for hybridoma preparation,and truncated Cap(tCap)with the NLS deletion,was used to screen the positive hybridoma.One hybridoma was obtained and analyzed by Western blotting,IFA and Immuno-EM.Results have shown that the hybridoma can recognize PCV2 virus,PCV2 VLP and linear Cap protein.3.NLS mediates PCV2 Cap protein nuclear export through phosphorylation.PCV2 Cap protein and its derivates were expressed via the baculovirus expression system,and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry.Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import,and a phosphorylation site(S17)was identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS)in the NLS domain to regulate Cap protein nuclear export.Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy.Moreover,the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells.Phosphorylation of NLS regulating Cap protein nuclear export provided more detailed knowledge of the PCV2 viral life cycle.In summary,PCV2 d VLP and anti-PCV2 VLP hybridoma were prepared and characterized in current study,NLS was demonstrated to be necessary and sufficient for PCV2 Cap protein nuclear export,and NLS regulated PCV2 Cap protein nuclear export by phosphorylation.