Role Of Ubiquitin-Proteasome System In The Porcine Circovirus Type2Infection In Vitro

Porcine circovirus type2(PCV2), a single-strand DNA virus, is the primary causative agent of porcine circovirus-associated diseases (PCVAD). The main target cells of PCV2are considered to be macrophage/monocyte-lineage cells and other antigen-presenting cells. The PCV2-associated PMWS is characterized as lymphoid depletion and histiocytic replacement in lymphoid tissues, which is associated with swine immunosuppressive or immunodepressive diseases and pave the way for secondary infection by other bacteria and virus, results in enormous economic losses to the world pig industry.Ubiquitin-proteasome system (UPS) is the major protein degradation pathway in eukaryotic cells. Except to degradation the bulk of cellular proteins, the UPS plays an important role in a variety of biological processes including apoptosis, cell division, cell cycle progression, MHC I molecule-mediated antigen processing, signaling pathways, inflammatory response, and protein processing. As the UPS has a vital role in many fundamental cellular processes, many viruses have reprogrammed the machinery according to their needs, such as viral replication (entry, transcription, translation, and release), viral immune evasion, cancer progression, diseases development. But the role of ubiquitin-proteasome system in the PCV2infection remains unknown.In the present study, we first identified the differentially expressed proteins in the PCV2-infected porcine alveolar macrophages. We then focus on the role of the ubiquitin-proteasome system on the PCV2replication and the differently expressed proteins of UPS-associated on the pathogenesis of PCV2. The main studies are as follows:1. Validation of differentially expressed proteins in the proteomic analysis of porcine alveolar macrophages infected with porcine circovirus type2Our previous study on the proteomic analysis of porcine alveolar macrophages infected with porcine circovirus type2and screened successfully32differentially expressed proteins. Base on the results, we validate the differentially expressed proteins through relative quantitative real-time PCR, western blot analysis, and confocal immunofluorescence assay. A total of21proteins which included9proteins with a change>2and12proteins with a change between1.5and2were validated to be differentially expressed in PCV2-infected PAMs. The identified proteins are involved in cytoskeleton organization, macromolecular biosynthesis, signal transduction, stress response, ubiquitin-proteasome system, and metabolic enzymes. The data obtained in our study may give us insight into the pathogenesis of PCV2infection.2. Inhibition of UPS impairs the early stages of PCV2replicationAmong the21differentially expressed proteins, the UPS was chosen for subsequent study for their potential role in the pathogenesis of PCV2. We first explored the role of UPS on the PCV2replication, proteasome inhibitors (MG132and Lactacystin) were used in the study. The results showed that treatment with MG132and lactacystin inhibited the DNA copies of PCV2in the PK15cells. To further identify which step(s) in the virus lifecycle are targeted by proteasome inhibitors, we first quantified the viral DNA copies after applying the inhibitors at different time points and found that inhibition of viral replication by the proteasome inhibitors is likely not dependent on the blockade of viral entry into the host cells. We further observed the effects of MG132or lactacystin on viral protein expression and RNA transcription and found the inhibitors significantly reduced the transcription and expression of ORF2. Taken together, these results indicate that the proteasome inhibitors decrease the number of viral DNA copies through suppression of viral protein expression and RNA transcription at the early infection stage.3. Proteasome inhibitors induce G2/M phase arrestAs the smallest DNA virus that could infect mammals, PCV2replication depends on cellular enzymes expressed during the S phase of cells growth. Experimental evidence shows that the UPS plays an important role in the regulation of the cell cycle, including the induction of S, G2/M arrest, and apoptosis. To investigate whether the downregulation of the PC V2growth is due to the effect of proteasome inhibitors on cell cycle progression, a flow cytometric analysis was performed on the PCV2-infected PK15cells treated with proteasome inhibitors. The results showed that MG132and lactacystin had no effect on the apoptosis and S phase but prolonged the G2/M phase in the dose-dependent manner, suggesting that inhibition of the UPS suppress the replication of PCV2in a cell cycle-dependent manner.4. PCV2infection may be associated with protein ubiquitinationThere are two successive steps involved in protein degradation: the target protein substrates are ubiquitinated through their covalent attachment to ubiquitin, and ubiquitinated proteins are degraded in the26S proteasome and release free ubiquitin. To elucidate the role of the UPS in PCV2infection, we investigated the protein ubiquitination and proteasome activity in PK15cells after PCV2infection and found that PCV2infection accumulated protein ubiquitination but had no effect on the chymotrypsin-like activity of proteasome. In addition, the viral DNA copies were reduced in the ubiquitin siRNA-transfected cells compared to the negative control. These results suggest that protein ubiquitination may be a critical process during the PCV2lifecycle.5. PSME1involves in the inflammatory response induced by PCV2infectionPSME1, a subunit of proteasome activator28, which was necessary for PA28to bind to and activate the proteasome, participates in the generation of the peptides that bind to newly synthesized MHC I molecular. In the present study, PSME1did not affect the viral DNA copies of PCV2but increase the expression of ORF2and activate the NF-κB signaling pathway which was validated by the up-regulation of inflammatory cytokines including IL-6, IL-8, TNF-a. After PCV2infection, although the NF-κB signaling pathway was preserved activation, its downstream cytokines were down-regulated. These results suggest that PSME1may be associated with immune evasion involved in PCV2infection, and the interaction between PSME1and PCV2impaired the up-regulation of PSME1and PCV2on the inflammatory cytokines.6. PSMB2regulates PCV2replication via NF-κB signal pathwayPSMB2, proteasome subunit β type2, is one of the three catalytic subunits in the proteasome and exhibits trypsin-like activity. Overexpression of PSMB2increased the viral DNA copies and ORF2expression in the PK15cells within48hpi. To further explore the mechanism of PSMB2increase the PCV2replication, the role of PSMB2on the NF-κB signaling pathway was studied. Overexpression of PSMB2in PK15cells, PCV2infection activated the NF-κB signaling pathway through inducing IκBα degradation and nuclear translocation of p65. Furthermore, PCV2infection the PSMB2-transfected PK15cells also up-regulated the inflammatory cytokines, including IL-6, IL-8, and TNF-a. Additionally, BAY11-7082, the inhibitor of NF-κB signaling pathway, inhibited the increase of viral titer which was induced by PSMB2. Taken together, PSMB2regulates the replication of PCV2via NF-κB signaling pathway.Taken together, inhibition of the UPS suppresses PCV2replication at the early infection stage through the degradation of viral DNA copies, protein expression, and viral transcription in a cell cycle-dependent manner. Furthermore, PCV2infection the PSME1-tranfected-PK15cells led the inflammatory cytokines disorders, which may be associated with immune evasion involved in PCV2infection. And PSMB2regulates the replication of PCV2via NF-κB signaling pathway. These results illustrate an important role of UPS in the regulation of the early stages of PCV2replication and in the inflammatory response of PCV2infection.