Study On Function Of Endoplasmic Reticulum Stress In Pseudorabies Virus Infection

Pseudorabies(PR)is an infectious disease caused by Pseudorabies virus(PRV).The infected newborn piglets mainly show neurological symptoms.Adult swine often have inapparent infection.Infection of pregnant sows can lead to abortion,stillbirth,etc.Boars show reproductive problems.Many studies have shown that the continuous synthesis of accumulated viral proteinsduring the process of virus infection and replication,whichlead to endoplasmic reticulum stress and activate unfolded protein responses to restore the endoplasmic reticulum homeostasis.Obviously,the virus should use the endoplasmic reticulum as part of its replicating device,it is necessary to deal with the pressure response of the endoplasmic reticulum and the related pathways downstream,and some viruses have been reported to interfere with endoplasmic reticulum stress through different mechanisms.However,the interaction between PRV and endoplasmic reticulum stress in the process of PRV infection and replication is not yet clear.In this study,we detected the protein changes of phosphorylated PERK(P-PERK),phosphorylated eIF2a(P-eIF2α),GRP78 and PRV in PRV-infected PK-15 by western bolting,and found that the protein level of GRP78 and phosphorylated eIF2a were significantly reduced,whilethe protein level of p-PERK were not significantly changed,which indicated that PRV infection could inhibit endoplasmic reticulum stress.In order to further study the effect of endoplasmic reticulum stress on PRV replication,PK-15 cells were treated with thapsigargin(Tg),an activator of endoplasmic reticulum stress,for 2 h,and after that PRV was used to infect PK-15 cellsfor 24 h or 48 h,cell supernatants and pellets were collected.Western-bloting was used to detect the protein level of P-PERK,P-eIF2a,GRP78,LC3-Ⅱ and PRV proteins.The results showed that Thapsigargin can significantly increase the protein level of GRP78,P-eIF2β,and LC3-Ⅱ,but PRV proteins were significantly reduced.This result indicates that Tg can prevent the replication ofPRV in PK-15 cells by enhancing endoplasmic reticulum stress;and then plaque assay were used to further detects PRV levels in cell supernatants and the results show that thapsigargin can also significantly reduced the release of PRV virion at 48 hafter PRV infection.To further clarify how endoplasmic reticulum stress affects PRV replication,an eukaryotic expression plasmid termedFlag-GRP78 was constructed in this experiment and transfected into PK-15 cells for 48 h.The results showed that overexpression of GRP78 increased P-eIF2a levels and decreased PRV replication,which indicates that overexpression of GRP78 could inhibit PRV replication by enhancing endoplasmic reticulum stress.The above results provide new directions for further elucidating the mechanism of PRV infection and replication in host cells.