Expression and functional analysis of the porcine circovirus type 2 Rep and ORF3 proteins

• Main Authors: Wei Li Lin, 林維莉
• Other Authors: Chienjin Huang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/04865829915580224077

博士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 100 === Porcine circovirus type 2 (PCV2) is a member belongs to the genus Circovirus of the Circoviridae family, and closely associated with a disease syndrome in pigs described as “postweaning multisystemic wasting syndrome” (PMWS) now known as porcine circovirus diseases (PCVD). The genome of PCV2 is a single-stranded circular DNA, and contains three major open reading frames (ORFs). They are ORF1, the rep gene, which encodes the Rep proteins responsible for virus replication; and ORF2, the cap gene, which encodes the immunogenic capsid (Cap) protein; and ORF3 encodes the protein for induce cell apoptosis. Synthesis of Rep protein as the first stage after PCV2 infection, is essential to viral DNA replication. The non-pathogenic type 1 PCV (PCV1) Rep protein has been shown to process the nuclear localization signal (NLS) for protein transport into nucleus and has DNA binding activity for viral DNA replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the PCV2 Rep protein, the defined coding regions of rep gene were cloned, expressed and analyzed the DNA binding activity. The results demonstrated that the full-length Rep (N314) and the deletion mutants coding the N-terminal 110 amino acid residues had the ability to bind to the 22-mer dsDNA fragment of the PCV2 origin sequence minimal binding site (ori MBS) sequence. Furthermore, to determine the region responsible for nuclear localization, several eukaryotic expression plasmids containing various coding regions of Rep protein identical to those expressed in E. coli were constructed. At 48 h post-transfection of plasmids into PK15 cells, cell were fixed, analyzed by indirect immunofluorescence assay (IFA) using mouse immune serum against PCV2 Rep, and observed by florescent microscopy. The full-length Rep protein (N314) and recombinant deletion mutants with 110 amino acid at N-terminal of Rep protein were dominantly localized in the nucleus. Examination of the 110 amino acid (aa) residues of the N-terminal region of PCV2 Rep revealed three clusters of basic aa with homology to other NLS. These results suggest that the utmost 20 residues in the N-terminal region could sufficiently mediate nuclear import of fusion protein, confirming its role as a functional NLS. Recent report showed that PCV2 induced apoptosis in the cultured porcine kidney cell (PK-15) via a viral protein encoded by ORF3. Monocyte/macrophage lineage cells are the major target cells for PCV2 infection. The purpose of this study was to expressed PCV2 ORF3 protein and further analyzing the role of ORF3 in inducing apoptosis in porcine peripheral blood mononuclear cells (PBMC). The full length of PCV2 ORF3 gene DNA fragment was amplification by PCR and suclone onto pET24 plasmid for preparation of the mouse antiserum against recombinant ORF3 protein. To analyze whether ORF3 is able to induce apoptosis, transient expression of the full length and the N- or C-terminal half of ORF3 protein in porcine PBMC was performed. After transfection, the transfected cells were detected by IFA and apoptosis induced by transient expression of viral protein was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling (TUNEL-labeling) which detected the DNA breakage and caspase activity. Quantification of TUNEL-positive cells showed that the percentage of apoptotic cells transfected with pcDNA/ORF3 was significantly higher the C-terminal half of ORF3 retained its ability to induce apoptosis similar to the full length of ORF3. Caspase activity assays demonstrated significant activation of caspase-3, -8, and -9 by the full length and the C-terminal half of ORF3 protein. The results suggest that nuclear localization may be required for apoptosis induction and the C-terminal half region of ORF3 contains either N53-68 or N85-104 regions responsible for nuclear localization. In summary, the functional regions of PCV2 Rep protein responsible for DNA binding activity and nuclear localization appear to be the N-terminal 110 residue portion of the protein. Another nonstructural protein of PCV2, ORF3, is capable of triggering apoptotic response in porcine PBMC maybe associated with the NLS. The apoptotic activity is correlated with the nuclear localization of ORF3, and nuclear localization may play an important role of PCV2 viral protein function and virus life cycle.