Study On The Function Of Conserved Aromatic Amino Acid Residues In NLS-A Region Of PCV2 Cap Protein

Porcine circovirus type 2(PCV2)associated diseases(PCVAD)have caused severe economic losses to the global pig industry.PCV2 is one of single-stranded,circular DNA viruses with a compact and small genome.There are multiple open reading frames(ORFs)in the genome,of which two ORFs(ORF1 and ORF2)play the crucial role in virus replication and assembly.ORF1 codes for replication-related proteins(Rep and Rep');while ORF2 encodes the sole capsid protein(Cap),which is the main antigens of PCV2.PCV2 Cap contains a Nuclear Localization Signal(NLS)located at the N-terminal end of this protein.The NLS consisting of the first 41 residues are rich of positively-charged basic amino acid residues.In 2018,Wanting Yu et al.reported that the NLS has the function as a cell-penetrating peptide(CPP),and its first 17 amino acid residues(NLS-A)play a key role in this function[40].The NLS-A at the N-terminus of NLS contains two aromatic amino acid residues,which are relatively conserved: tyrosine(Y)at the third position and phenylalanine(F)or Y at the eighth position.No studies have reported the effect of both of the conserved residues of the NLS-A on the functions of the CPP and the proliferation of PCV2.In this project,PCV2 d strain was choosed as the research object,and alanine(A)residue was used to replace 3Y and 8F of the NLS-A.By synthesizing FITC-fluorescent labeled NLS-A polypeptide(FITC-NLS-A)and flow cytometry analysis,the effect of conserved aromatic amino acid residues in the NLS-A region on the function of NLS-A penetrating cells was studied.Meanwhile,infectious DNA clones of mutants and wild-type of PCV2 were constructed respectively.These PCV2 infectious DNA clones were transfected into both the Porcine Kidney-15(PK15)cell line and the intestinal porcine epithelial cell line(J2),respectively,and PCV2 viruses are successfully rescued.Furthermore,the function of the conserved aromatic amino acid residues on the replication of PCV2 was studied by comparing the replication kinetics in vitro of these rescued viruses between the mutants and the wild-type strains.The results will provide theoretical and experimental basis for further understanding of the replication and proliferation mechanism of PCV2.The results include:(1)The 8F at the N-terminus of the NLS-A can significantly improve the efficiency of cell penetration of the NLS-A(P<0.05),while,the 3Y also plays a positively role in the NLS-A penetrating the cells in a concentration-dependent manner.(2)The 3Y and 8F of the NLS-A can significantly improve the replication ability of PCV2 in vitro(P<0.01).Both of the mutants and wild-type strain of PCV2 have been successfully rescued via transfections of these infectious DNA clones into PK15 cells,and the rescued viruses(mutants and wild type)are capable of proliferation in vitro and consecutively passaging for more than 10 generations,respectively in the cells,which was confirmed by PCR and real-time PCR by using cell-free culture supernatants of each generation.Immunofluorescence assays(IFAs)also support the conclusion above.Moreover,the genomic DNA copy numbers of the mutants are all significantly lower than that of wild type strains(P<0.01).In addition,the results of TCID50 through inoculating the rescued viruses(mutants and wild type)from the 1st to 5th generations to the negative PK15 cell cultures revealed that the mutants all had significantly lower titer than that of the wild type(P<0.01).Thus,it can be concluded that the conserved aromatic amino acid residues in the NLS-A can significantly enhance the replication ability of PCV2 in vitro(P<0.01).(3)The functions of conserved aromatic amino acid residues in the NLS-A region is independent of cells types.Similarly,in the PK15 cells,these viruses have been successfully rescued from the infectious DNA clones(mutants and wild type),respectively,in J2 cells and also the rescued viruses are able to be consecutively passaged at least 10 gengerations,respectively.However,the genome copy numbers of the mutantare all significantly lower than that of the wild type(P<0.01).Altogether,this study suggests that both conserved aromatic amino acid residues in the NLS-A of the PCV2 Cap play crucial roles in PCV2 replication and cell-penetration in vitro.In addition,the function of the NLS-A is independent of cell types,which provided theoretical and experimental basis for further understanding the mechanism of the NLS of the PCV2 Cap,the replication and proliferation mechanism of PCV2.