Study On Peste Des Petits Ruminants Virus Nucleocapsid Protein And C Protein Regulate Autophagy Via Inducing Endoplasmic Reticulum Stress

Peste des petits ruminants(PPR)an acute,febrile and contact infectious disease,PPR virus(PPRV)is the causative of PPR which mainly infects goats and sheep.Causing a great loss to husbandry due to its high morbidity and mortality of PPR.Adverse conditions such as hypoxia,pathogen infection,and calcium imbalance can cause massive accumulation of unfolded proteins and misfolded proteins in the endoplasmic reticulum(ER),resulting in ER stress(ERS).Autophagy is a protective mechanism in eukaryotes that help to recover cellular homeostasis by degrading the virus and its parts,senescence organelles after viral infection.Studies have shown that viruses regulate autophagy by inducing ERS to benefit viral replication.The previous studies have demonstrated that PPRV induce autophagy and ERS to promote virus replication in caprine endometrial epithelial cells(EECs),but the relationship between PPRV-induced ERS,autophagy and PPRV replication is still unclear.Focusing on this question,we made an exploration:1.Effects of PPRV-induced ERS on autophagy in EECs.The ERS inhibitor 4-PBA were treated in PPRV-infected EECs.Immunoblotting analysis showed that the expression of LC3-II(the biomarker of autophagy)was significantly down-regulated,while GRP78(the biomarker of ERS)was inhibited during PPRV infection in EECs;Laser confocal microscopy assay showed that 4-PBA significantly reduced the number of LC3 punctate aggregations in the cytoplasm of PPRV-infected EECs.Conversely,TG were treated(ERS activator)in the PPRV-infected cells,the expression of LC3-II significant up-regulation.Further it also significantly increased the number and fluorescence intensity of LC3 punctate aggregations.These results showed that PPRV-induced ERS positively regulates autophagy.2.PERK/eIF2α pathway regulates PPRV-induced autophagy.GSK2606414(GSK)were treated to reduce the activation of the PERK/eIF2α pathway after PPRV infection.Immunoblotting assays showed that GSK treatment reduced the phosphorylation levels of PERK,meanwhile,the expression of LC3-II was also significantly down-regulated.It suggested that the PERK/eIF2α pathway activated by PPRV was involved in the regulation of autophagy.Furthermore,small interference RNAs(si RNAs)were adopted to block the expression of PERK,eIF2α and ATF4 respectively.Results showed that the expression of LC3-II,ATG5,ATG7 and HSP70 were significantly inhibited which along with the down-regulation of the protein expressions of the PERK,eIF2α and ATF4.In conclusion,the PERK,eIF2α and ATF4 may involve in upregulating the expression of ATG5,ATG7,HSP70 and LC3-II.In this way,PERK/eIF2α pathway was involved in facilitating autophagy,and benefiting PPRV replication.3.The N and C proteins of PPRV regulate autophagy by activating the PERK/eIF2αpathway.First,Immunoblotting was adopted to detect the PERK/eIF2α pathway and autophagy-related molecular after exogenously expressing the PPRV N protein and C protein in EECs respectively.The research results showed that the N protein and C protein of PPRV promoted the expression of GRP78(biomarker of ERS)and p-eIF2α,ATF4(key molecular of PERK/eIF2α pathway),furthermore,significantly up-regulated the expression of ATG5,ATG7,and LC3-II.Data demonstrated that PPRV N protein and C protein are involved in activating PERK/eIF2α pathway and facilitating autophagy.4-PBA were adopted to block ERS that induced by viral N protein or C protein,data showed that 4-PBA significantly impaired the expression of LC3-II.It demonstrated that N protein and C protein are involved in ERS-mediated autophagy.GSK were adopted to block the PERK/eIF2α pathway that activated by N protein and C protein,data showed that the expressions of ATG5,ATG7,and LC3-II were significantly down-regulated.Research results above demonstrated that N protein and C protein activated the PERK/eIF2αpathway to facilitate autophagy.In conclusion,PPRV infection facilitate autophagy by inducing ERS in EECs.The N protein and C protein of PPRV are involve in upregulating autophagy and benefiting PPRV replication via the activation of PERK/eIF2α pathway.This study deepened the understanding of the pathogenic mechanism of PPRV,and provided a theoretical basis for the prevention and treatment of PPR.