Identification Of The Sub-cellular Localization Signal Of Porcine Epidemic Diarrhea Virus N Protein

Porcine epidemic diarrhea virus（PEDV）is the etiological agent of entero-pathogenic diarrhea inswine.The disease is characterized by enteritis, vomiting, watery diarrhea and even dehydration.Theprevalence of Porcine epidemic diarrhea（PED）all over the world has resulted heavy economic losses tothe swine industry. One of the most abundant viral proteins produced inside a coronavirus-infeccted cellis the nucleocapsid（N）protein, has an important roles in modulating virus replication and altering hostcell processes. So, the study of identify sub-cellular localization signals of porcine epidemic diarrheavirus N protein were carried out, which will provide reliable informations for developing novel vaccineand designing antiviral strategy, and for understanding of the pathogenic mechanism of PEDV.In the present study, to identify whether there were potential sub-cellular localization signal inPEDV N protein, we first conducted a bioinformatic analysis of the protein using existing motifprediction algorithms. PSORT Ⅱ indicated that PEDV N protein contained a pat7motif（261PKKNKSR267）,To investigate whether this and other unknown signals operated to determine thesub-cellular localization of N protein, we divided the protein into three regions based on amino acidsequence feature of coronavirus N protein and combinations of regions1plus2and2plus3and clonedthese downstream of green fluorescent protei（nGFP）generating plasmids pAcGFP-NR1、pAcGFP-NR2,pAcGFP-NR3, pAcGFP-NR1+2and pAcGFP-NR2+3. Vero E6cells were co-transfected withpAcGFP-NR1, pAcGFP-NR2, pAcGFP-NR3, pAcGFP-NR1+2and pAcGFP-NR2+3andpDsRed2-B23.1, this latter construct allowing the expression of a nucleolar marker protein, B23.1,tagged to DsRed2. Confocal analysis the sub-cellular localization characteristics of N protein and otherregions at24h post-transfection. The live cell imaging indicated that AcGFP and DsRed2uniformlylocalized to both the cytoplasm and nucleus, but not the nucleolus, whereas AcGFP-N protein localizedto both the cytoplasm and nucleolus but not the nucleus. pAcGFP-NR1+2protein same to AcGFP-Nand localized to the cytoplasm and nucleolus, pAcGFP-NR2+3protein was predominantly cytoplasmicin localization, pAcGFP-NR1protein localized predominantly to the nucleolus, pAcGFP-NR2proteinlocalized predominantly to the cytoplasm and appeared also to accumulate in the nucleolus to the lowlevel as the cytoplasm, whereas AcGFP-NR3localized predominantly to the cytoplasm.This datasuggested that a potential nucleolus localization signals （NoLS） could be located in region1, thatregion2contained a potential nuclear export signal（NES）and also a possible NoLS, but we proposethat the NoLS in region1is necessary and sufficient to direct N protein to the nucleolus. Interestingly,region2also contained one predicted NLS, which could either have been submissive to the NES or notfunctional. The data also suggested that region3contained a potential NES because region3wasdirected to the cytoplasm. In order to finely identify the sub-cellular localization signals, a series ofexpression constructed containing fragments of region1,2,3were constructed, and in the same way VeroE6cells were transfected or co-transfected with plasmid pDsRed2-B23.1, and in order to better displaythe results at the time of identify the NES, the DAPI was used to sign nucleus. Finally, the cells werefixed at24h post-transfection for confocal analysis by direct fluorescence. This results suggested that a NoLS （71SNWHFYYLGTGPHGDLRYRT90） located in region1and a NES（325LAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNV364）located in region3werepreliminary identified, and a functional NES（222LVAAVKDALKSLGI235）was also identified in region2. It is an important foundation to further finely identify functional sub-cellular localization signals.Various studies have showed that N protein of coronavirus can localize to both the cytoplasm andnucleus/nucleolus, and RNA viruses interact with the nucleolus to usurp host-cell functions and recruitnucleolus proteins to facilitate virus replication.To preliminary identify the interaction of PEDV Nprotein and fibrillarin in Vero E6cells, the fibrillarin gene were amplified by RT-PCR from Vero E6cells with a pairs of primers synthesized according to human fibrillarin gene, and cloned into eukaryoticexpression vector pDsRed2-N1to generate the recombinant plasmid pDsRed2-Fib. Vero E6cells weretransfected with plasmids pAcGFP-N and pDsRed2-Fib. Western blot results showed that the fusionproteins expressed in transfected Vero E6cells. Furthermore, the PEDV N protein and the fibrillarinshowed co-localization features in nucleolus of co-transfected Vero E6cells through confocalmicroscopy observation. The results would be helpful to elucidate the interaction of PEDV N proteinand nucleolus proteins.The nucleolus phosphoprotein B23.1is identified as a high-level phosphoprotein in granularregions of the nucleolus, this protein can as a shuttle protein in protein nuclear import, Furthermore,B23.1protein possesses molecular chaperoning activities. Previously research indicated that thenucleolus both in terms of gross morphology and protein content was altered in infectious bronchitiscoronavirus-infected cells. To understand the effect of PEDV N protein on B23.1protein in Vero cellduring infection with the PEDV, Vero E6cells were transfected with recombinant plasmidpDsRed2-B23.1, after12、18and24h of transfection, observe the changes of B23.1protein use confocalmicroscopy analysis.Results indicate that the PEDV N protein and the B23.1phosphoprotein showedco-localization features in co-transfected cells and B23.1protein to begin changes at12h after infectedPEDV even crush with time extension, contrast control group that have light changes. These resultsstrongly suggested viruses interact with the nucleolus to usurp host-cell functions and recruit nucleolusproteins to facilitate virus replication.