Differential Proteome Analysis Of PK-15 Cells Infected With Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2), a member of the family Circoviridae, is a small, non-enveloped virus, with a circular, single-stranded DNA genome. Three ORFs of PCV2 have been identified. ORF1 encodes a replication-associated protein (Rep), ORF2 encodes the capsid protein (Cap), and ORF3 encodes ORF3 protein. PCV2 can induce the immune suppression of pig. In this study, we analyze the differential expression proteins in PCV2-infected PK15 cells and protein interaction between PCV2 and host cellular protein. The aim is to investigate the mechanism of replication and infection of PCV2.Construction of infectious clone of PCV2In field conditions, pigs can be infected simultaneously by different subtypes of PCV2. The pure PCV2 virus was needed to investigate the pathogenesis of PCV2 virus subtype. In this study, to obtain the pure virus stock of PCV2 TZ, we cloned the whole genome of PCV2 TZ. Subsequently, the infectious clone of PCV2 TZ were constructed to rescue the virus of PCV2 rTZ and then measure the culture features of the rescued PCV2 rTZ. Results showed that the rescued virus of PCV2 rTZ possessed the similar replication ability compared with the wild-type virus. In this study, the purified virus stock of PCV2 rTZ can provide a basis for further investigation.Differential proteome analysis of PK-15 cells infected with PCV2To analyze the differential proteome of PK-15 cells infected with PCV2, the rescued PCV2 rTZ was used to infect PK-15 cells, and two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. From the results of 2-DE analysis, we found 61 differential protein spots, which appeared majority at 48-96 h after PCV2 infection. There are 44 protein spots were successfully identified using the methods of MALDI-TOF/TOF mass spectrometry, including 22 up-regulated protein spots and 22 down-regulated protein spots.22 up-regulated protein spots represent 18 proteins, which are actins, tubulins, cytokeratins, acidic ribosomal phosphoprotein P0, tyrosyl-tRNA synthetase, peroxiredoxin 6, peroxiredoxin 4, vacuolar proton pump subunit B 2, proteasome subunit p40, NUP43 protein, the ras-related protein Rap-2c, prohibitin, protein phosphatase 2 isoform 8, arginine deiminase, pyruvate dehydrogenase E1 component subunit beta, and isocitrate dehydrogenase. In addition, 22 down-regulated protein spots represent 22 protein spots, which are T-plastin, intermediate filament ptoteins, heat shock protein beta-1, elongation factor 2, replication protein A2,32kDa, purine nucleoside phosphorylase, GMP synthetase, hypoxanthine-guanine phosphoribosyltransferase, histidine triad protein member 5, transaldolase, galactose mutarotase, peptidylprolyl isomerase D, alpha enolase, 6-phosphogluconolactonase, phosphoglucomutase 3,3'-phosphoadenosine 5'-phosphosulfate synthase, proteasome beta 3 subunit, proteasome activator complex subunit 1, suppressor of G2 allele of SKP1, SH2/SH3 adaptor protein, homeobox prospero-like protein PROX1. The up-regulated proteins associated with cytoskeleton, biosynthesis, lipoprotein metabolism, ubiquitin proteasome pathway, signal transduction, and gene regulation. The down-regulated protein associated with protein translation and elongation, RNA processing and biosynthesis, carbohydrate degradation, amino acid transport and metabolism, the ubiquitin-proteasome pathway, signal transduction, and gene regulation. Moreover,20 corresponding genes of the differential proteins were quantified by real time RT-PCR to validate the transcriptional profiles. Results showed that transcriptional profiles of these 20 protein were partial identical with 2-DE analysis. Commercial mAbs were used to further analyze protein profiles of beta-actin, alpha-tubulin, and cytokeratin 8 during PCV2 infection. Results showed protein profiles of alpha-tubulin, and cytokeratin 8 were consistent with the 2-DE analysis. In summary, PCV2 infected PK-15 cells changed the cellular cytoskeleton associated proteins, stress response associated proteins, biological synthesis associated proteins, ubiquitin proteasome pathway associated proteins, signal transduction and gene regulation associated proteins.Identification of PCV2 Cap protein with the cellular protein a-tubulinSubcellular localization of virus-encoded protein indicates the replication place of virus. In order to analyze the subcellular localization of PCV2 Rep and Cap protein, the eukaryotic expression vector of Rep and Cap were respectively constructed to transfect PK-15 cells, and the rescued PCV2 rTZ were utilized to infect PK-15 cells. The results showed that Rep protein of PCV2 localized in the nucleus of PK-15 cells at 24 h p.i. and 48 h, p.i. and some can be found in the cytoplasm at the 72 h p.i. Cap protein of PCV2 localized in the nucleus at 24 h p.i., and some can be found in the cytoplasm at 48 h p.i, and 72 h p.i. Therefore Rep and Cap protein could be translocated in PK-15 cells. The cytoskeleton is responsible for cargo transport in cells. Based on the differential proteome analysis, the cytoskeleton associated proteins were changed enormously. So the methods of double immunofluorescence and co-precipitation were utilized to analyze the association between beta-actin, alpha-tubulin, cytokeratin 8 proteins and the PCV2-encoded protein. From the results we found that only Cap protein of PCV2 co-locolized with the alpha-tubulin. Further co-immunoprecipitation results showed that cellular a-tubulin form the compound with Cap protein in PCV2 infected PK-15 cells. All of results demonstrate that Cap protein of PCV2 interacts with the cellular protein a-tubulin.