Study Of The Biological Characteristics Of Rep And ORF3 Protein Of Duck Circovirus

DuCV is a single-stranded DNA virus without envelope, a member of genus Circovirus in the family Circoviridae. Studies indicated that infection of DuCV mainly caused lymphocyte depletion, necrosis, and histiocytosis in the bursa of Fabricius of ducks. Our previous fluorescence in situ hybridization test showed DuCV could cause obvious lesions in the spleen, thymus and Bursa of Fabricius, suggesting that the pathogenesis of DuCV may be related to the apoptosis of lymphocyte, which is quite similar to PCV2. Previously, we verified that the Cap protein of DuCV possessed nuclear localization signal(NLS), but there is no final conclusion on whether Rep protein has NLS, and no study was done to indentify the pro-apoptosis function of the Rep of two genotypes. We found ORF3 has pro-apoptosis function in our earlier study, but it is still need to be verified that whether there is distinction between the ORF3 of the two genotypes.The study was done as follows:1. Inverstigation of the NLS of the two genotypes of Rep protein of DuCVTwo atypical NLSs were found in Rep1 by online analysis of cNLS Mapper software(http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_ Mapper_form.cgi), NLS1 was located in N terminal 10KRWVFTINNPTFEDYVHVLEFCTLDNCK37, NLS2 was located in C terminal,244ITSNKEPRDWYKSEFDLSALYRRINKYLVYN274, while there was only one atypical NLS, NLS1, was calculated in Rep2. Four pairs of RFP fusion expression plasmids were constructed to express Rep1 and Rep2 according to the analysis of the software, namely, wild type plasmids(Rep1-DsRed2, Rep2-DsRed2), NLS1-eliminated plasmids(Rep1?N10-37-DsRed2, Rep2?N10-37-DsRed2), NLS2-eliminated plasmids(Rep1?N244-274-DsRed2,Rep2?N244-274-DsRed2), NLS1 and NLS2-eliminated plasmids(Rep1?N10-37?N244-274-DsRed2, Rep1?N10-37?N244-274-DsRed2). By observation through laser scanning confocal microscope after tranfecting H1299 cells for 48 h, it is showed that the elimination of NLS2 in C terminal did not change the nuclear localization function, while after the elimination of NLS1, Rep1 and Rep2 lost the ability to transfer to the cell nuleus and were distributed in cytoplasm, which verified that the NLS in N terminal was indispensable for the Rep protein transffering.2. Study of the pro-apoptosis functions of the two genotypes of Rep protein of Du CVTwo pairs of recombinant plasmids for expressing the two genotypes of Rep protein were constructed with flag tag(p3×Flag-DuCV1-Rep、p3×Flag-DuCV2-Rep) and EGFP tag(pEGFP-DuCV1-Rep、pEGFP-DuCV2-Rep), the pro-apoptosis functions of Rep protein was tested by Annexin V-FITC Detection Kit, PE Annexin V Detection Kit and APODERECT Detection Kit. For lack of appropriate duck-originated cell lines, considering the distinction of pro-apoptosis function of Rep protein in heterogeneous cell lines, the ananlysis of pro-apoptosis function was conducted in mammal cell line CHO and avian-originated cell line DF-1.During the detection of apoptosis, the adherent cells and mixture of adnerent and supernate cells were collected respectively at 24, 48 and 72 hpt for the crossed analysis of pro-apoptosis function of Rep protein. The result showed that the Rep protein of the two genotypes both possessed certain pro-apoptosis function. Compared to Rep1, pro-apoptosis function of Rep 2 is weaker, and the function appeared relatively later in CHO cells,indicating the mild pro-apoptosis function of Rep2. It is also found that DF-1 is more sensitive to Rep2, and the pro-apoptosis function is easier to be detected.3. Detection of the distinction of the pro-apoptosis functions of the two genotypes of ORF3 protein of DuCVTwo pairs of recombinant plasmids for expressing the two genotypes of ORF3 protein were constructed with flag tag(p3 × Flag-DuCV1-ORF3 、 p3 × Flag-DuCV2-ORF3) and EGFP tag(pEGFP-DuCV1-ORF3 、 pEGFP-DuCV2-ORF3), the pro-apoptosis function of ORF3 protein was tested in CHO cell lines and DF-1cell lines by Annexin V-FITC Detection Kit, PE Annexin V Detection Kit and APO-DERECT Detection Kit. During the detection of apoptosis, the adherent cells and mixture of adnerent and supernate cells were collected respectively at 24, 48 and 72 hpt for the crossed analysis of pro-apoptosis function of ORF3 protein. The result showed that the two genotypes of ORF3 protein both possess certain proapoptosis functions. The ORF3 of DuCV2 appeared obvious pro-apoptosis function which upgraded firstly and then descended over time. The ORF3 of DuCV1 presented pro-apoptosis function later which upgraded gradually as time goes on, while in the later stage, the proapoptosis function is significantly more active than DuCV2. Compared to avian-originatedcell DF-1, the pro-apoptosis function of ORF3 appeared later in mammal CHO cell line,indicating that ORF3 of DuCV have differnt pro-apoptosis functions to different cell lines.4. Study of the distinction of Caspase activities induced by the two genotypes of ORF3 and Rep proteinThe activity of caspases is the typical feature of apoptosis. In this study, the real-time RT-PCR methods for detecting mRNA of caspase 3, caspase 8 and caspase 9 were established.The plasmids expressing the two genotypes of ORF3 were transfected into CHO cells respectively, and real-time RT-PCR was carried at 24, 48 and 72 hpt. The results showed the m RNA of caspase 3 and caspase 8 increased at different time after the transfection of the plasmids of the two genotypes, with no significant differnce from control group. The plasmids expressing the two genotypes of Rep and ORF3 protein were transfected into CHO cells respectively, and the activity of caspase 3, caspase 8 and caspase 9 were tested with Caspase-3/8/9 Colorimetric Assay Kit at 24, 48 and 72 hpt. The result showed at 72 hpt, the activity of caspase 3 in Rep1 group was significantly higher than control group and Rep2 group(p<0.01),and the activity of caspase 3 in ORF3-DuCV1 group was significantly higher than control group and ORF3-DuCV2 group(p<0.05). At other time points, the level of the three apoptosis proteins has no significant distinction with control group. The results showed that the ORF3 and Rep protein of DuCV may play pro-apoptosis function by other caspase-independent apoptosis pathways.