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Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

ABSTRACT: Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death, and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3 was. PCV1ORF3-associated cell death was caspase dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase 1 (PARP) cleavage; however, whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids contain a domain capable of inducing cell death, whereas the C terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not determined or not solely determined by the ORF3 protein.

SUBMITTER: Chaiyakul M

PROVIDER: S-EPMC2953190 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

Chaiyakul Mark M Hsu Karolynn K Dardari Rkia R Marshall Frank F Czub Markus M

Journal of virology 20100901 21

Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties o ...[more]

PMID: 20810737

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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.

Project description:Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes located within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the cytotoxicity of C. difficile may be modulated by genomic variants outside the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between cytotoxicity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model cytotoxicity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci and changes in cytotoxicity. Combined, these data support a model of C. difficile disease wherein cytotoxicity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections, including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Finally, we highlight the intersection of loci identified by the GWAS for different phenotypes and clinical severity. This strategy to identify overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCE Clostridioides difficile has two major disease-mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study, we demonstrate via multiple approaches that genomic variants outside the PaLoc are associated with changes in cytotoxicity. These genomic variants may provide new avenues of exploration in the hunt for novel disease-modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical for clinical infection, such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and, as such, vary in their appropriateness for certain bacterial genome-wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important for both bacterial function and human clinical disease.

Dataset Information

Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

Publications

Cytotoxicity of ORF3 proteins from a nonpathogenic and a pathogenic porcine circovirus.

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