Functional Analysis Of Putative ORF5 Protein Of Porcine Circovirus Type 2 And Antiapoptotic Mechanism Of Viral ORF4 Protein

Porcine circovirus type 2(PCV2),the smallest virus known to infect mammals, has been recognized as the primary aetiological agent of porcine circovirus-associated diseases(PCVAD) in swine. PCV2, as a nonenveloped virus, is classified in the genus Circovirus of the family Circoviridae and its genome is a single-stranded, closed circular, ambisense, unsegmented DNA. The virus is predicted to possess 11 overlapping open reading frames(ORFs), of which four have been characterized in detail. ORF1 encodes the two replicases Rep and Rep’, ORF2 encodes the capsid protein Cap, ORF3 encodes an apoptotic protein called ORF3 protein, and ORF4 encodes an antiapoptotic protein called ORF4 protein(its antiapoptotic mechanism is still unclear). Sequence alignment analysis of PCV2 isolates from varying geographic regions suggested that the putative ORF5 gene(proposed to exist by Hamel et al) might not encode a functional protein, as translation of the protein was found to terminate after only seven amino acids in some Chinese PCV2 strains. However, several groups have aligned the genomic sequences of various PCV2 isolates and found a region of extremely high identity(over 95%) at the amino acid level, suggesting that PCV2 encodes a novel protein, although different names for this possible protein have been used in the literature(Gen Bank accession no. O92288, Q77GS3, Q77S04, etc). Therefore, it is necessary to investigate the expression and function of the putative ORF5 protein during PCV2 infection, and clarifying the molecular mechanism of ORF4 protein in regulating apoptosis will be also helpful for us to understand the PCV2 pathogenesis. The obtained results are shown as follow:(1) Confirmation of the expression of PCV2 ORF5 protein at both the transcriptional and translational level. Porcine alveolar macrophages 3D4/2(PAMs) infected with wild-type PCV2 Yangling strain(wPCV2) at the indicated time points were subjected to RT-PCR and Northern blot analyses. The results demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. In addition, immunoreactive band with the predicted size was detected in the protein sample from PAMs transfected with pEGFP-ORF5 and simultaneously IFA signal was found in PAMs infected with wPCV2 by using our homemade mouse anti-ORF5 polyclone antibodies(pAbs), including the pAbs prepared from BALB/c mice immuned with GST-ORF5 fusion proteins, peptide containing the potential epitope of ORF5 protein synthesized by chemical synthesis or eukaryotic expression plasmid pcDNA-ORF5, respectively. Thus, expression of the ORF5-encoded protein exists at both the transcriptional and translational level within PCV2-infected cells. These data establish the foundation for further research on the characteristics and functions of the putative ORF5 protein. However, due to the inability to detect the putative PCV2 ORF5 protein by western blot, additional studies are required to validate the expression of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn.(2) Functional analysis of GFP-fused ORF5 Protein. To analyze the function of ORF5-encoded protein, an ORF5-deficient PCV2 infectious DNA clone(PCV2Δ) was constructed. Replication kinetics of PCV2Δ indicated that the putative ORF5 protein is not essential for PCV2 replication in cell culture. Real-time quantitative RT-PCR analysis of the expression curves of viral ORF1, ORF2, ORF3, ORF4 and ORF5 mRNAs demonstrated that PCV2 ORF5 gene initiates transcription during the early stage of infection and peaks at 48 h post-infection(hpi), and the site-directed mutation within ORF5 does not affect its transcription, suggesting that the ORF5 gene may initiate transcription at a more upstream site. Additionally, the expression levels of ORF1 and ORF2 transcripts in PCV2Δ-infected cells were significantly lower than those measured in w PCV2- or rPCV2-infected cells from 12 hpi to 72 hpi, suggesting that the lack of ORF5 reduced the transcriptional capacity of ORF1 and ORF2 during the first 72 hours after infection. To further clarify the role of ORF5 protein plays in the process of PCV2 infection, a eukaryotic expression plasmid pEGFP-ORF5 was constructed. PAMs transfected with pEGFP-ORF5 were used to examine the impact that the putative ORF5 protein has on the physiological functions of cells. The obtained results demonstrated that the GFP-fused ORF5 protein is degraded via the proteasome, inhibits PAM growth, prolongs the S-phase of the cell cycle, localizes to the endoplasmic reticulum(ER) and induces ER stress, activates NF-κB and stimulates the upregulation of NF-κB-mediated downstream genes. Nevertheless, due to the possibility that functions of GFP-ORF5 perhaps not completely represent those of ORF5 alone, additional experiments are needed to corroborate the function of the ORF5 protein during PCV2 infection in vitro.(3) Identification of cellular proteins that interact with the PCV2 ORF4 and ORF5 proteins. To screen for cellular partners of PCV2 ORF4 and ORF5 proteins, ORF4 and ORF5 proteins were used as bait in a yeast two-hybrid approach to screen a cDNA library derived from the porcine alveolar macrophages of a healthy pig. A total number of four porcine proteins(including FHC, SNRPN, COX8 A and Lamin C) were found to interact with the PCV2 ORF4 protein and five porcine proteins(including GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were identified as cellular partners of the PCV2 ORF5 protein.(4) Study on the molecular mechanism of the PCV2 ORF4 protein antagonizing apoptosis. PCV2 ORF4 protein has previously been proposed as an anti-apoptotic protein, but how this protein plays a role in antagonizing apoptosis is still unclear. To clarify this issue, ferrtin heavy chain(FHC) was chosen for further characterization. Results of GST Pull-down and co-IP indicated that ORF4 protein was capable of binding FHC in intracellular and extracellular at the protein level, and results of confocal microscopy demonstrated that ORF4 protein co-localized with FHC in the cytoplasm. All these data suggested that PCV2 ORF4 protein interacts with the FHC protein. Due to the important role of FHC in apoptosis reportedly was dose dependent, lentivirus-mediated FHC overexpression(LV-FHC) and knockdown(sh-FHC) indicated that FHC is indeed involved in PCV2-induced apoptosis. To further investigate the correlation between FHC expression and the inhibition of PCV2-induced apoptosis, The resulting stably transfected cell lines expressing GFP-ORF4 fusion protein and ORF4-deficient PCV2 mutant virus(PCV2Δ) were used to demonstrate that PCV2 ORF4 protein has no effect on the transcription of FHC gene but reduces the density of bioactive FHC in PCV2-infected cells by some way of protein modification, and thereby antagonizes apoptosis.To sum up, this study elucidated that PCV2 ORF5 protein is not essential for PCV2 replication, but the GFP-fused ORF5 protein inhibits cell proliferation via prolongation of the S-phase of the cell cycle, localizes to the endoplasmic reticulum(ER) and induces ER stress, activates NF-κB and stimulates the upregulation of NF-κB-mediated downstream genes. In addition to above findings, we also uncovered that PCV2 ORF5 protein interacts with GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3, ORF4 protein interacts with FHC, SNRPN, COX8 A and Lamin C, and PCV2 ORF4 protein antagonizes apoptosis via stabilizing concentration of ferritin heavy chain(FHC) through physical interaction.