Abstract
dsRNA-dependent protein kinase R (PKR) is an interferon-inducible protein that mediates antiviral effects and induces apoptosis. We studied PKR-related apoptosis mechanisms by transfecting wild type pcDNA-carp-wtPKR, a catalytically inactive mutant pcDNA-mut-carpPKR, and empty plasmid in Epithelioma papulosum cyprini (EPC) cells, designated wtPKR, mutPKR, and pcDNA3.1, respectively. PKR was inefficiently expressed from wtPKR unlike mutPKR that produced high PKR levels detected by western blot. eIF2α phosphorylation increased in wtPKR-transfected cells, while for mutPKR, phosphorylation was not different from non-transfected controls. Flow-cytometry revealed high level of apoptosis in wtPKR transfected cells, corresponding with high cytopathic effect. mutPKR and pcDNA3.1 transfection gave significantly less apoptosis and were not different from each other. Caspase-8 and -9 were activated for wtPKR, suggesting death receptor-caspase-8 and mitochondrion-dependent caspase-9 activated pathways, similar to mammalian cells. These findings suggest that the induction of apoptosis via the caspase-8 and -9 pathways are conserved in vertebrate taxa and likely play a role in viral infections of lower vertebrates.
Highlights
Type I interferon (IFN) response is the major innate immune defense mechanism against viral infection
The best known function of the activated protein kinase R (PKR) is the control of protein translation via phosphorylation of the alpha subunit of eukaryotic initiation factor 2, whose main function is inhibition of protein synthesis in order to prevent viruses from producing new progeny [9]. eIF2α is the primary substrate of PKR and its phosphorylation correlates with the induction of programmed cell death that renders it to serve as a molecular determinant of apoptosis [10]
We find that PKR overexpression in Epithelioma papulosum cyprini (EPC) cells induces apoptosis following eIF2α phosphorylation, and activation of caspase-8 and -9
Summary
Type I interferon (IFN) response is the major innate immune defense mechanism against viral infection. Production of type I IFN is stimulated by recognition of invading viruses through different host sensors [1]. Induction of different IFN-stimulated genes (ISGs) leads to the establishment of an antiviral state in host cells [4]. The dsRNA activated protein kinase R (PKR) is an ISG constitutively expressed in most mammalian cells and is activated by the binding of dsRNA to dsRNA-binding motifs (dsRBMs) at its N-terminus [5,6,7]. The best known function of the activated PKR is the control of protein translation via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α), whose main function is inhibition of protein synthesis in order to prevent viruses from producing new progeny [9]. PKR has been identified, cloned, and characterized in several fish species such as olive flounder (Paralichthys olivaceus) [11], zebrafish (Danio rerio) [12], crucian carp (Carassius carassius) [13], and rock bream
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