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The Porcine Circovirus Type 2 Nonstructural Protein ORF3 Induces Apoptosis in Porcine Peripheral Blood Mononuclear Cells.

ABSTRACT: Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To analyze whether the PCV2 nonstructural protein ORF3 is able to induce apoptosis in nature target cells, transient expression of ORF3 in porcine peripheral blood mononuclear cells (PBMC) was performed, and apoptosis was confirmed by terminal dexoynucleotidyl transferase (TdT)-mediated BrdUTP-nick end labeling (TUNEL) assay. The apoptotic responses induced by the full length or the C-terminal half of ORF3 were significantly higher (p < 0.001) than that of cells transfected with the control plasmid. In contrast, the N-terminal half of ORF3 restrictively localized in the cytoplasm and remarkably reduced its ability to induce apoptosis, the apoptotic activity might be correlated with the nuclear localization of ORF3. Furthermore, two clusters of basic residues on the C-terminal half region at the amino acid residues 53-68 and 85-104 could mediate the nuclear localization of fusion protein, confirming their potential role as a nuclear localization signal.

SUBMITTER: Lin WL

PROVIDER: S-EPMC3249664 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The Porcine Circovirus Type 2 Nonstructural Protein ORF3 Induces Apoptosis in Porcine Peripheral Blood Mononuclear Cells.

Lin Wei-Li WL Chien Maw-Sheng MS Wu Pei-Ching PC Lai Chen-Li CL Huang Chienjin C

The open virology journal 20111213

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To analyze whether the PCV2 nonstructural protein ORF3 is able to induce apoptosis in nature target cells, transient expression of ORF3 in porcine peripheral blood mononuclear cells (PBMC) was performed, and apoptosis was confirmed by terminal dexoynucleotidyl transferase (TdT)-mediated BrdUTP-nick end labeling (TUNEL) assay. The apoptotic responses induced by the full length or the ...[more]

PMID: 22216074

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Project description:Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316-323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.

Dataset Information

The Porcine Circovirus Type 2 Nonstructural Protein ORF3 Induces Apoptosis in Porcine Peripheral Blood Mononuclear Cells.

Publications

The Porcine Circovirus Type 2 Nonstructural Protein ORF3 Induces Apoptosis in Porcine Peripheral Blood Mononuclear Cells.

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