Study Of The Mechanism Of HSV-1 Facilitates Its Replication By Regulating Inositol-requiring Enzyme 1(IRE1)Stress Pathway

Endoplasmic reticulum(ER)plays important roles in viral replication.Viral infection can lead to endoplasmic reticulum stress response(ER stress),The ER stress is marked by the activation of a series of signaling pathways,called the unfolded protein response(UPR),which consists of three distinct signal pathways,PRKR-like ER kinase(PERK),inositol-requiring enzyme 1(IRE1)and activated transcription factor 6(ATF6).IRE1 has two isoforms in mammalian cells:IRE1α and IRE1β.In this paper,we study the relationship between the HSV-1 replication and RNase,kinase activity of IRE1α.We also study the possibility of the regulation of IRE1α by viral protein.1.IRE1α mRNA and protein expression are upregulated byHSV-1 infection at late stage postinfection.The RNase activity of IRE1α signaling branch has been the focus of investigation for the relationship between IRE1α and HSV-1 infection.We investigate the effect of HSV-1 infection on IRE1α signaling pathway by the expression of IRE1αmRNA and protein expression level.We found that HSV-1 infection results in the upregulating of IRE1α mRNA and phosphorylated protein.We also found that it is necessary to maintaining IRE1α expression for HSV-1 replication.2.Activation of kinase activity and inhibition of RNase activity of IRE1α during HS V-1 infection.IREα is the most conserved signal pathway in cells from yeast to humans,which is a transmembrane protein containing the kinase/endoribonuclease(RNAse)activity,both residing on its cytosolic face.We further investigate the effect of viral replication on RNase activity.Firstly,we found that RNase activity of IRE1αmaintain constant during viral infection,which is consist with others research results.Secondly,we use the drugs which are the regulator of RNase activity of IRE1α and found that the RNase activity of IRE1α is inhibited by viral infection.We confirm this result by XBP1s overexpression and ERAD inhibition.We detect the relationship between viral infection and kinase activity of IRE1αand found that HSV-1 viral replication is inhibited by both mutation and inhibitor of kinase activity of IRE la,which indicated that kinase activity is necessary for viral replication.Research has showed that JNK activity is induced by IRE la kinase activity.We also found that JNK signaling pathway is inhibited by both siIRE1α and the inhibition of kinase activity of IRE1α.We suppose that it is necessary for viral replication to active IRE la signaling pathway because IRE la kinase activity can result in activation of JNK signaling pathway.3.IRE la is regulated by HSV-1 ICPO and gD.Since IRE1α stress signaling pathway is manipulated by viral replication,we want to investigate the viral proteins by which regulating the IRE1α RNase/kinase activity.ICPO is a multifunctional protein that associates with a large number of cellular proteins involved in transcription,cell cycle regulation,DNA repair and the interferon response.It is reported that ICPO is necessary for the induction of E and L gene expression during productive infection.We detect whether ICPO could regulate the activity of IRE1αa.We found that RNase activity of IRE1α is increased by siICP0,which suggested that IREla RNase activity is inhibited by ICPO during viral productive infection.However,we have not found ICPO co-localized with IREla in cells.We supposed that IRE1α RNase activity is regulated by ICPO by some unknown mechanisms,which need to be further investigated.Virion associated gD interacts with cellular proteins to trigger virion membrane fusion while newly synthesized gD uses the same interactions to directly interfere with subsequent virion entry or by inducing the endocytosis of cell surface receptors.Just as gD expression,JNK activation induced by kinase of IRE1α is also activated at later infection.We detect the relationship between gD protein and kinase activity of IRE1α.As shown above,viral replication is inhibited by siIRE1α.We also found that HSV-1 gD co-localized with IRE1α in cells.In the conclusion,we suppose that IRE 1-JNK signaling is activated by gD expression to facilitate viral infection.