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.

No abstract

Background: Current vaccines against Japanese encephalitis virus (JEV) of flaviviruses have some disadvantages, such as the risk of virulent reversion. Non-structural protein NS1 is conserved among flaviviruses and confers immune protection without the risk of antibody-dependent enhancement (ADE). Therefore, NS1 has become a promising vaccine candidate against flaviviruses. Methods: A NS1-based vaccine (LTB-NS1 Δ63 ) with a truncated NS1 protein (NS1 Δ63 ) fused to E. coli heat-labile enterotoxin B subunit (LTB) was expressed in E.coli and explored for its ability to induce immune responses. Safety of LTB-NS1 Δ63 was assessed by determining its toxicity in vitro and in vivo. Protective capability of LTB-NS1 Δ63 and its-induced antisera was evaluated in the mice challenged with JEV by analyzing mortality and morbidity. Findings: LTB-NS1 Δ63 induced immune responses to a similar level as LTB-NS1, but more robust than NS1 Δ63 alone, particularly in the context of oral immunization of mice. Oral vaccination of LTB-NS1 Δ63 led to a higher survival rate than that of NS1 Δ63 or live-attenuated JEV vaccine SA14À14À2 in the mice receiving lethal JEV challenge. LTB-NS1 Δ63 protein also significantly decreases the morbidity of JEV-infected mice. In addition, passive transfer of LTB-NS1 Δ63 -induced antisera provides a protection against JEV infection in mice. Interpretation: NS1 Δ63 bears JEV NS1 antigenicity. Besides, LTB-NS1 Δ63 could serve as a novel protein-based mucosa vaccine targeting JEV and other flaviviruses.