Differential Proteome Analysis of Host Cells Infected with Porcine Circovirus Type 2

Abstract

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which is an emerging swine immunosuppressive disease. To uncover cellular protein responses in PCV2-infected PK-15 cells, the comprehensive proteome profiles were analyzed utilizing two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification. Multiple comparisons of 2-DE revealed that the majority of changes in protein expression occurred at 48-96 h after PCV2 infection. A total of 34 host-encoded proteins, including 15 up-regulated and 19 down-regulated proteins, were identified by MALDI-TOF/TOF analysis. According to cellular function, the differential expression proteins could be sorted into several groups: cytoskeleton proteins, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation. Western blot analysis demonstrated the changes of alpha tubulin, beta actin, and cytokeratin 8 during infection. Colocalization and coimmunoprecipitation analyses confirmed that the cellular alpha tubulin interacts with the Cap protein of PCV2 in the infected PK-15 cells. These identified cellular constituents have important implications for understanding the host interactions with PCV2 and brings us a step closer to defining the cellular requirements for the underlying mechanism of PCV2 replication and pathogenesis.

Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

“…Our previous report identified the PCV2 Cap protein bound to ␣-tubulin in a coimmunoprecipitation assay (28,73). In this study, we demonstrated that Cap also colocalized and interacted with the microtubule-associated dynein IC1 chain.…”

“…PCV2 is the smallest known virus, and the virion binds to heparan sulfate and chondroitin sulfate B glycosaminoglycans (GAGs) on the cell surface (24)(25)(26) before entering the cell via clathrin-mediated endocytosis in monocytic cells or via an actinand small-GTPase-dependent pathway in epithelial cells (27). Recent research showed that the PCV2 Cap protein binds to ␣-tubulin in PCV2-infected cells (28), and the Hsp70/Cap complex activated the NF-B pathway and reduced caspase-3 activity in PCV2-infected monocytic cells (29). However, the mechanism of intracellular transport of the PCV2 virion remains unclear.…”

“…Common immunoblotting (IB) was performed as previously described (28). Briefly, cells were lysed in lysis buffer after infection or other treatments for the indicated times.…”

“…The supernatant was incubated with immunoprecipitation (IP) antibody at 4°C for 8 h, and immune complexes were precipitated by incubation with fresh agarose for another 8 h at 4°C. Beads were washed five times with PBS, and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting as previously described (28). In the coimmunoprecipitation assay, the IP antibodies used were mouse anti-Cap MAb, mouse anti-Flag M2 MAb (F1804; Sigma), and rabbit anti-Myc pAb (R1208-1; Huaan Biological Technology).…”

Interactions of porcine circovirus 2 with its hosts

“…During PCV2 infection, the expression levels of cellular genes are extensively modulated, and the resulting differentially expressed proteins are involved in the cytoskeleton, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation, and immune response [38][39][40][41][42][43][44][45]. The virus-host interactions related to PCV2 proliferation are shown in Fig.…”