Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

中文翻译：

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日本脑炎病毒通过激活PERK途径诱导凋亡和脑炎。

积累的证据表明，日本脑炎病毒（JEV）感染会触发内质网（ER）应激和神经元凋亡。据报道，ER应激传感器蛋白激酶R样内质网激酶（PERK）可以在急性或长期ER应激下诱导细胞凋亡。但是，PERK在JEV诱导的细胞凋亡和脑炎中的确切作用仍然未知。在这里，我们报道JEV感染在体外和体内均激活PERK-ATF4-CHOP细胞凋亡途径，PERK激活还促进应激颗粒的形成，进而抑制JEV诱导的细胞凋亡。但是，PERK抑制剂可降低细胞凋亡，表明JEV激活的PERK主要通过PERK-ATF4-CHOP细胞凋亡途径诱导细胞凋亡。在已报道可诱导内质网应激的JEV蛋白中，只有JEV NS4B可以诱导PERK激活。据报道，PERK通过二聚作用形成活性分子。免疫共沉淀试验表明，NS4B与PERK相互作用。此外，甘油梯度离心显示NS4B诱导PERK二聚化。NS4B中的LIG-FHA和LIG-WD40结构域都需要诱导PERK二聚化，这表明JEV NS4B通过同时通过不同的基序与它们相互作用来将两个PERK分子拉在一起。PERK失活可减少JEV感染期间的脑细胞损伤和脑炎。此外，JEV NS4B的表达足以在小鼠中通过PERK诱发脑炎，这表明JEV主要通过其NS4B激活PERK引起脑炎。综上所述，我们的发现为JEV引起的脑炎提供了新颖的见解。重要事项日本脑炎病毒（JEV）感染会触发内质网（ER）应激和神经元凋亡。据报道，ER应激传感器蛋白激酶R样内质网激酶（PERK）可以在急性或长期ER应激下诱导细胞凋亡。然而，ER应激反应的PERK途径是否在JEV诱导的细胞凋亡和脑炎中起重要作用尚不清楚。在这里，我们发现JEV感染会激活神经元细胞和小鼠大脑中的ER压力传感器PERK。在JEV感染后，PERK激活通过PERK-ATF4-CHOP细胞凋亡途径诱导细胞凋亡。在JEV蛋白prM，E，NS1，NS2A，NS2B和NS4B中，只有NS4B激活PERK。此外，活化的PERK参与JEV和NS4B诱导的细胞凋亡和脑炎。这些发现为JEV引起的脑炎提供了一种新颖的治疗方法。