Effects Of Endoplasmic Reticulum Stress Induced By Peste Des Petits Ruminants Virus On Virus Replication

Pete des petits ruminants(PPR)is an acute or subacute disease in small ruminants,the infectious disease is caused by the petite des petits ruminants virus(PPRV)of the genus Morbillivirus of the Paramyxoviridae family.PPRV mainly infects small ruminants including wild animals and is listed as legally reported animal diseases by the Office International Des Epizooties(OIE).After the host body is infected with PPRV,the clinical symptoms mainly include fever,necrotizing stomatitis,diarrhea,and bronchopneumonia.The mortality rate of PPR is 50% to 80%.PPR caused huge economic losses to the animal husbandry industry.The endoplasmic reticulum is the important place for protein synthesis,assembly and maturation in cells,and it is also a necessary place for the virus to complete its own life cycle.During the process of virus maturation and replication,the host cell's endoplasmic reticulum needs to be used to synthesize a large number of viral proteins,resulting in the accumulation of a large number of unfolded or misfolded proteins,which causes endoplasmic reticulum stress(ERS),and activates the unfolded protein response(UPR).Most RNA viruses can cause endoplasmic reticulum stress,but there is no research report on PPRV can induce endoplasmic reticulum stress.To investigate whether PPRV can induce endoplasmic reticulum stress after infecting host cells and the molecular mechanism of endoplasmic reticulum stress regulating its replication,the research contents and results of this project are as follow:(1)PPRV infection induces endoplasmic reticulum stress in Caprine endometrial epithelial cells(EECs).In this study,the morphological structure of the endoplasmic reticulum of EECs was observed by transmission electron microscopy.Our found that PPRV(MOI = 3)infection caused significant swelling of the endoplasmic reticulum cavity.After EECs were infected with PPRV,the expression of GRP78 and GRP94,the marker molecules of endoplasmic reticulum stress,were significantly increased in protein and m RNA expression by immunoblotting and q RT-PCR detection,indicating that PPRV infection can induce endoplasmic reticulum stress in EECs.This indicates that PPRV infection can induce endoplasmic reticulum stress in host cells.In order to explore which signal pathways of UPR activated by PPRV infection induced endoplasmic reticulum stress,the key factors on the three signal pathways of UPR(PERK,ATF6,IRE1)were detected by immunoblotting and quantitative PCR,we found PPRV infection of EECs promotes the phosphorylation of PERK and e IF2? protein and the expression levels of ATF4 and CHOP m RNA,indicating that the PERK-e IF2? pathway is activated;while the ATF6 and IRE1 pathways are not activated.Toanalyze the effects of various viral proteins on endoplasmic reticulum stress,we transfected EECs with plasmids containing viral P,C,H,V,and N genes.The results showed that HA-C and HA-N proteins significantly increased the expression of GRP78 and GRP94,indicating that the C and N proteins of PPRV play an important role in the induction of endoplasmic reticulum stress in EECs.(2)Effects of endoplasmic reticulum stress on PPRV replication.To further analyze the role of endoplasmic reticulum stress and the UPR pathway in the replication of PPRV,we used TG,4-PBA,GSK2606414,and si RNAs that interfered with GRP78,GRP94,and PERK to promote or inhibit endoplasmic reticulum stress.The virus titer and the expression of N protein were tested.The results showed that the endoplasmic reticulum stress inducer TG promoted the replication of PPRV at 24 h,but inhibited the replication of PPRV at 48 h and72 h.4-PBA alleviated the replication of PPRV after alleviating endoplasmic reticulum stress,indicating that Endoplasmic reticulum stress promotes PPRV replication to a certain extent;the use of the drug GSK or interference with PERK to inhibit the PERK pathway reduces PPRV replication.The study also found that interfering with the expression of GRP94 molecule had no significant effect on viral replication,while interfering with another marker molecule GRP78 significantly inhibited the replication of PPRV.To sum up,this study found that PPRV infection of EECs can induce endoplasmic reticulum stress and activate the PERK pathway.PPRV C and N proteins are the key proteins for PPRV to induce endoplasmic reticulum stress in host cells.ERS can promote the replication of PPRV,PPRV can use the PERK pathway and GRP78 to enhance its replication.The research results laid a theoretical foundation for further exploration of the pathogenic mechanism of PPRV.