Regulation Of Endoplasmic Reticulum Chaperones GRP78 And GRP94 In PRV Infection And Replication

The endoplasmic reticulum(ER)is a vital organelle which is involved in cell metabolism,calcium homeostasis,and the synthesis,folding and trafficking of proteins.The process of virus reproduction will accumulate a large number of viral proteins in endoplasmic reticulum and finally induce endoplasmic reticulum stress(ERs).Unfolded protein response(UPR)is a series of adaptive responses to alleviate endoplasmic reticulum stress.The UPR regulates the replication of virus at the meanwhile.The endoplasmic reticulum chaperones are induced in the process of UPR.They are mainly responsible for folding,assembly and transportation of proteins,but not for their composition.This function is of great significance for accelerate expression of viral protein.Pseudorabies virus(PRV)can cause Aujeszky’s disease in swine and other animals in many countries.Natural host and reservoir of PRV is swine,leading to massive economic losses worldwide.There have been many achievements about PRV,but little was known about the internal immune regulation of PRV infection.Hence,the interaction between ERs and PRV infection and the regulating effects of two endoplasmic reticulum molecular chaperones(GRP78 and GRP94)on PRV replication were explored in this work.The research contents are shown as follows:1.Interaction between endoplasmic reticulum stress and replication of PRVThe morphological changes of ER in BHK-21 cells after PRV infection was observed by transmission electron microscopy to explore the influence of PRV infection on the ER.At the same time,GRP78 expression was enhanced at different time spots after PRV infection.These results confirmed that the ERs would be induced during PRV infection.Then The influences of ER stress activator,Tg and ER stress inhibitor,TUDCA on PRV propagation were explored by measuring the progeny viral titers.And the titers of progeny virus increased with the severity of ER stress.These results confirmed that the intrinsic connection between the PRV-infection and ER stress.2.The regulatory effect of GRP94 on PRV replicationBHK-21-GRP94 cells and BHK-21-GRP94-KO cells were constructed by recombinant lentiviral vector-aided expression and CRISPR/Cas9 system.After PRV infection,it was found that viral titer was significantly enhanced in BHK-21-GRP94 cells,but viral titer could not be impacted in BHK-21-GRP94-KO cells.In order to explore the specific causes of this phenomenon,UPR-related proteins,including GRP78,GRP94,ATF6 and e IF2α were detected by Western blot.It was found that the expression level of GRP78 were enhanced significantly in BHK-21-GRP94-KO cells which was infected by PRV.It was speculated that the host cell might regulate the protein expression to increase the expression of GRP78 to make up for the lack of GRP94.3.The regulatory effect of GRP78 on PRV replicationRNA interring technique was used to inhibit the expression of GRP78 in BHK-21 cells.The effect of GRP78 on PRV replication was similar to that of GRP94.Similarly,after measuring UPR-related proteins,it was found that the expression of GRP94 was also slightly up-regulated after inhibiting GRP78,but it was not significant.Surprisingly,ATF6 pathway was triggered in BHK-21-GRP78-siRNA cells after PRV infection,because GRP78 has much more function and cannot be compensated by GRP94 alone.The inhibition of GRP78 can be better compensated by the expression of target gene of ATF6 pathway afterward.4.Interaction between GRP78,GRP94 and PRV structural proteinsIn order to further explore the role of the two chaperones in virus replication,eight viral lipid membrane glycoproteins were transient expressed in BHK-21 cells.The interactions between GRP78,GRP94 and viral glycoproteins were examined by immunoprecipitation technique.Finally,the results showed that GRP78 interacted with four glycoproteins,and GRP94 interacted with three glycoproteins.This result confirmed that those two chaperones could bind with a variety of glycoproteins to accelerate the folding and assembly of viral proteins,and then affect the efficiency of virus replication.