Abstract
In this study, the mechanism of Muscovy duck reovirus (MDRV) p10.8 protein-induced pathogenesis was investigated, with a focus on endoplasmic reticulum (ER) stress. In chicken embryo fibroblasts cell lines (DF1), pCI-neo-flg-p10.8 protein transfection increased the phosphorylation (p-) levels of PERK and eIF2α as shown by Western blotting analysis and led to the dissociation of BiP from PERK as shown by co-immunoprecipitation (Co-IP) analysis. Results of treatment with both ER stress activator and inhibitor further confirmed that p10.8 protein induced ER stress. Subsequently, using flow cytometry analysis, it was also found that p10.8 protein induced cell cycle arrest during the G0/G1 phase. Furthermore, p10.8 transfection increased the phosphorylation levels of PERK and eIF2α, and reduced the expression levels of CDK2, CDK4, and Cyclin E according to Western blotting analysis. Treatment with ER stress activator and ER stress inhibitor after p10.8 protein transfection in DF1 cells further indicated that p10.8 protein induced ER stress, which resulted in cell cycle arrest. The results of knockdown of either PERK or eIF2α genes further confirmed that p10.8 protein-induced ER stress led to cell cycle arrest through the PERK/eIF2α pathway. Further results showed that p10.8 protein induced ER stress and apoptosis in DF1 cells. The expression levels of p-PERK, p-eIF2α, CHOP, cleaved-Caspase12, and cleaved-Caspase3 were increased by p10.8 protein. Test results of treatment with each of Tunicamycin, TUDCA and knockdown of PERK, and eIF2α, confirmed that p10.8 protein induced ER stress involving apoptosis via the PERK/eIF2α pathway. In conclusion, MDRV p10.8 protein induced ER stress that caused cell cycle arrest and apoptosis through the PERK/eIF2α pathway.
Highlights
Ducklings infected with the reovirus were first reported in 1950 (Kaschula, 1950), and Muscovy duck reovirus (MDRV) was first isolated in 1972 (Gaudry et al, 1972)
Protein Co-IP analysis showed that p10.8 protein caused binding immunoglobulin protein (BiP) to dissociate from protein kinase RNA-like ER kinase (PERK) and increased the phosphorylation levels of PERK and eIF2α, which led to the progression of endoplasmic reticulum (ER) stress in DF1 cells (Figures 1G–J)
Studies examining the spleen transcriptome profile of Muscovy ducklings in response to MDRV indicate that this reovirus could induce innate immune function through four signal pathways: the Janus kinase-signal transducer, activator of transcription signaling pathway (JAKSTAT), the retinoic acid-inducible gene I (RIG-I)-like, and Tolllike receptor (TLR) signaling pathways (Wu et al, 2017b)
Summary
Ducklings infected with the reovirus were first reported in 1950 (Kaschula, 1950), and Muscovy duck reovirus (MDRV) was first isolated in 1972 (Gaudry et al, 1972). MDRV p10.8 Protein Function (Cai et al, 2015). Polypeptides undergo protein folding in the endoplasmic reticulum (ER). If they are not accurately folded, they are degraded or re-folded in the ER. Sometimes the unfolded polypeptides accumulate in the ER and cause ER stress (Rashid et al, 2015). Cells promote the unfolded protein reaction (UPR). UPR is initiated and mediated by three transmembrane stress sensors, protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1), and activating transcription factor 6 (ATF6) (Schönthal, 2012; Han and Kaufman, 2016; Kroeger et al, 2018). Lead to the accumulation of unfolded proteins in ER, such as pathogenic infection, which activates the sensors by dissociating the BiP (Kopp et al, 2018)
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