The IRE1 Arm Of The Unfolded Protein Response Regulates Inflammatory Response Induced By PRV Variant Strain Infection

Since 2011,severe pseudorabies virus(PRV)outbreaks associated with highly virulent,antigenic-variant strains have occurred on many pig farms in China,the virus is a member of the alphaherpesvirus subfamily,resulting in serious economic losses for the pig industry worldwide.Previous studies have shown the PRV JS-2012 variant strain caused higher mortality and severe pathological lesions in pigs,as compared with a classic PRV SC strain,and the Bartha-K61 vaccination cannot provide complete protection against challenge with variant PRV strain in pigs.However,how the variant strain induced higher motality and pathological lesions remains controversial.The endoplasmic reticulum(ER)stress is a common consequence in herpesvirus infection that plays an important role in virus-induced inflammatory response,which can induce highly inflammatory cytokines expression by activating the unfolded protein response(UPR).Here,we showed that a mechanism for PRV-triggered inflammatory response,which is regulated by the ER stress pathway.Firstly,to determine whether PRV strains infection induce inflammatory cytokines expression in vitro,we measured the expression of major cytokines(IL-6,IL-8,TNF-?)after PRVs infection in pig kidney cells(PK-15)by qPCR and ELISA.The expression of IL-6,IL-8 and TNF-? was up-regulated along with prolonged infection time,peaking at 36 hr p.i.Importantly,the PRV variant strain(JS-2012)induced higher mRNA and protein expression of IL-6,IL-8,TNF-? than classical virulent PRV strain(SC)in the late stage of viral infection(36hr p.i.),which did not caused by different virus replication level.Secondly,we demostrated that both the infected PRV variant and classical strains induced ER stress and triggered UPR in PK-15 host cells.Both of them can activate three arms of UPR,among them,the infection of classic strain and variant strain can up-regulate the expression of each signal molecule in the PERK pathway,but the activation of each molecule by the two strains is not significantly different.Then,we verified the ATF6 pathway,the results showed that the infection of both strains can activate the cleavage of ATF6,but cannot upregulate the expression of its downstream signaling molecules Calreticulin and Calnexin,it showed that the infection of two strains of PRV activates the incomplete ATF6 pathway.Importantly,compared with the classical strain,the variant strain can activate the phosphorylation level of the IRE1 molecule and the expression of its downstream signaling molecules XBP1 s and EDEM,the results indicated that the classical strain and the variant strain have significant differences in the degree of activation of the IRE1 pathway.To further investigated whether the activatedd ER stress is responsible for the high expression of inflammatory cytokines induced by PRVs infection,we pretreated PK-15 cells with the chemical chaperone 4-phenylbutyric acid(4-PBA),which was used to alleviate the ER stress.The 4-PBA pretreatment reduced the inflammatory cytokines(IL-6,IL-8,TNF-?)expression in two kinds of PRV strains infected-cells.Further studies show that IRE1 regulates the PRV variant strain-induced inflammatory response and positively infects the PRV variant strain replication,as shown by functional studies with siRNA and chemical inhibitors.Overall,results in this study demostrated that IRE1 arm of UPR plays an important role in PRV variant strain-caused severe inflammatory response and higher mortality and provide new insights into the pathogenic mechanism of PRV and the base for novel strategies to develop the vaccine to protect mammals from PRV.